Lipid-mediated polynucleotide administration to reduce likelihood of subject&#39;s becoming infected

ABSTRACT

A method for delivering an isolated polynucleotide to the interior of a cell in a vertebrate, comprising the interstitial introduction of an isolated polynucleotide into a tissue of the vertebrate where the polynucleotide is taken up by the cells of the tissue and exerts a therapeutic effect on the vertebrate. The method can be used to deliver a therapeutic polypeptide to the cells of the vertebrate, to provide an immune response upon in vivo translation of the polynucleotide, to deliver antisense polynucleotides, to deliver receptors to the cells of the vertebrate, or to provide transitory gene therapy.

This application is a continuation of U.S. patent application Ser. No.08/187,630, filed Jan. 26, 1994, now U.S. Pat. No. 5,703,055, which is adivisional of Ser. No. 07/496,991, filed Mar. 21, 1990, now abandonedwhich is a continuation in part of Ser. No. 07/467,881, filed Jan. 19,1990, abandoned, which is a continuation in part of Ser. No. 07/326,305,filed Mar. 21, 1989, abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to introduction of naked DNA and RNAsequences into a vertebrate to achieve controlled expression of apolypeptide. It is useful in gene therapy, vaccination, and anytherapeutic situation in which a polypeptide should be administered tocells in vivo.

Current research in gene therapy has focused on “permanent” cures, inwhich DNA is integrated into the genome of the patient. Viral vectorsare presently the most frequently used means for transforming thepatient's cells and introducing DNA into the genome. In an indirectmethod, viral vectors, carrying new genetic information, are used toinfect target cells removed from the body, and these cells are thenre-implanted. Direct in vivo gene transfer into postnatal animals hasbeen reported for formulations of DNA encapsulated in liposomes and DNAentrapped in proteoliposomes containing viral envelope receptor proteins(Nicolau et al., Proc. Natl. Acad Sci USA 80:1068-1072 (1983); Kaneda etal., Science 243:375-378 (1989); Mannino et al., Biotechniques 6:682-690(1988). Positive results have also been described with calcium phosphateco-precipitated DNA (Benvenisty and Reshef Proc. Natl. Acad Sci USA83:9551-9555 (1986)).

The clinical application of gene therapy, as well as the utilization ofrecombinant retrovirus vectors, has been delayed because of safetyconsiderations. Integration of exogenous DNA into the genome of a cellcan cause DNA damage and possible genetic changes in the recipient cellthat could predispose to malignancy. A method which avoids thesepotential problems would be of significant benefit in making genetherapy safe and effective.

Vaccination with immunogenic proteins has eliminated or reduced theincidence of many diseases; however there are major difficulties inusing proteins associated with other pathogens and disease states asimmunogens. Many protein antigens are not intrinsically immunogenic.More often, they are not effective as vaccines because of the manner inwhich the immune system operates.

The immune system of vertebrates consists of several interactingcomponents. The best characterized and most important parts are thehumoral and cellular (cytolytic) branches. Humoral immunity involvesantibodies, proteins which are secreted into the body fluids and whichdirectly recognize an antigen. The cellular system in contrast, relieson special cells which recognize and kill other cells which areproducing foreign antigens. This basic functional division reflects twodifferent strategies of immune defense. Humoral immunity is mainlydirected at antigens which are exogenous to the animal whereas thecellular system responds to antigens which are actively synthesizedwithin the animal.

Antibody molecules, the effectors of humoral immunity, are secreted byspecial B lymphoid cells, B cells, in response to antigen. Antibodiescan bind to and inactivate antigen directly (neutralizing antibodies) oractivate other cells of the immune system to destroy the antigen.

Cellular immune recognition is mediated by a special class of lymphoidcells, the cytotoxic T cells. These cells do not recognize wholeantigens but instead they respond to degraded peptide fragments thereofwhich appear on the surface of the target cell bound to proteins calledclass I major histocompatibility complex (MHC) molecules. Essentiallyall nucleated cells have class I molecules. It is believed that proteinsproduced within the cell are continually degraded to peptides as part ofnormal cellular metabolism. These fragments are bound to the MHCmolecules and are transported to the cell surface. Thus the cellularimmune system is constantly monitoring the spectra of proteins producedin all cells in the body and is poised to eliminate any cells producingforeign antigens.

Vaccination is the process of preparing an animal to respond to anantigen. Vaccination is more complex than immune recognition andinvolves not only B cells and cytotoxic T cells but other types oflymphoid cells as well. During vaccination, cells which recognize theantigen (B cells or cytotoxic T cells) are clonally expanded. Inaddition, the population of ancillary cells (helper T cells) specificfor the antigen also increase. Vaccination also involves specializedantigen presenting cells which can process the antigen and display it ina form which can stimulate one of the two pathways.

Vaccination has changed little since the time of Louis Pasteur. Aforeign antigen is introduced into an animal where it activates specificB cells by binding to surface immunoglobulins. It is also taken up byantigen processing cells, wherein it is degraded, and appears infragments on the surface of these cells bound to Class II MHC molecules.Peptides bound to class II molecules are capable of stimulating thehelper class of T cells. Both helper T cells and activated B cells arerequired to produce active humoral immunization. Cellular immunity isthought to be stimulated by a similar but poorly understood mechanism.

Thus two different and distinct pathways of antigen processing produceexogenous antigens bound to class II MHC molecules where they canstimulate T helper cells, as well as endogenous proteins degraded andbound to class I MHC molecules and recognized by the cytotoxic class ofT cells.

There is little or no difference in the distribution of MHC molecules.Essentially all nucleated cells express class I molecules whereas classII MHC proteins are restricted to some few types of lymphoid cells.

Normal vaccination schemes will always produce a humoral immuneresponse. They may also provide cytotoxic immunity. The humoral systemprotects a vaccinated individual from subsequent challenge from apathogen and can prevent the spread of an intracellular infection if thepathogen goes through an extracellular phase during its life cycle;however, it can do relatively little to eliminate intracellularpathogens. Cytotoxic immunity complements the humoral system byeliminating the infected cells. Thus effective vaccination shouldactivate both types of immunity.

A cytotoxic T cell response is necessary to remove intracellularpathogens such as viruses as well as malignant cells. It has provendifficult to present an exogenously administered antigen in adequateconcentrations in conjunction with Class I molecules to assure anadequate response. This has severely hindered the development ofvaccines against tumor-specific antigens (e.g., on breast or coloncancer cells), and against weakly immunogenic viral proteins (e.g., HIV,Herpes, non-A, non-B hepatitis, CMV and EBV).

It would be desirable to provide a cellular immune response alone inimmunizing against agents such as viruses for which antibodies have beenshown to enhance infectivity. It would also be useful to provide such aresponse against both chronic and latent viral infections and againstmalignant cells.

The use of synthetic peptide vaccines does not solve these problemsbecause either the peptides do not readily associate withhistocompatibility molecules, have a short serum half-life, are rapidlyproteolyzed, or do not specifically localize to antigen-presentingmonocytes and macrophages. At best, all exogenously administeredantigens must compete with the universe of self-proteins for binding toantigen-presenting macrophages.

Major efforts have been mounted to elicit immune responses to poorlyimmunogenic viral proteins from the herpes viruses, non-A, non-Bhepatitis, HIV, and the like. These pathogens are difficult andhazardous to propagate in vitro. As mentioned above, synthetic peptidevaccines correspond to viral-encoded proteins have been made, but havesevere pitfalls. Attempts have also been made to use vaccinia virusvectors to express proteins from other viruses. However, the resultshave been disappointing, since (a) recombinant vaccinia viruses may berapidly eliminated from the circulation in already immune individuals,and (b) the administration of complex viral antigens may induce aphenomenon known as “antigenic competition,” in which weakly immunogenicportions of the virus fail to elicit an immune response because they areout-competed by other more potent regions of the administered antigen.

Another major problem with protein or peptide vaccines is anaphylacticreaction which can occur when injections of antigen are repeated inefforts to produce a potent immune response. In this phenomenon, IgEantibodies formed in response to the antigen cause severe and sometimesfatal allergic reactions.

Accordingly, there is a need for a method for invoking a safe andeffective immune response to this type of protein or polypeptide.Moreover, there is a great need for a method that will associate theseantigens with Class I histocompatibility antigens on the cell surface toelicit a cytotoxic T cell response, avoid anaphylaxis and proteolysis ofthe material in the serum, and facilitate localization of the materialto monocytes and macrophages.

A large number of disease states can benefit from the administration oftherapeutic peptides. Such peptides include lymphokines, such asinterleukin-2, tumor necrosis factor, and the intergerons; growthfactors, such as nerve growth factor, epidermal growth factor, and humangrowth hormone; tissue plasminogen activator; factor VIII:C;granulocyte-macrophage colony-stimulating factor; erythropoietin;insulin; calcitonin; thymidine kinase; and the like. Moreover, selectivedelivery of toxic peptides (such as ricin, diphtheria toxin, or cobravenom factor) to diseased or neoplastic cells can have major therapeuticbenefits. Current peptide delivery systems suffer from significantproblems, including the inability to effectively incorporate functionalcell surface receptors onto cell membranes, and the necessity ofsystemically administering large quantities of the peptide (withresultant undesirable systemic side effects) in order to deliver atherapeutic amount of the peptide into or onto the target cell.

These above-described problems associated with gene therapy,immunization, and delivery of therapeutic peptides to cells areaddressed by the present invention.

SUMMARY OF THE INVENTION

The present invention provides a method for delivering a pharmaceuticalor immunogenic polypeptide to the interior of a cell of a vertebrate invivo, comprising the step of introducing a preparation comprising apharmaceutically acceptable injectable carrier and a nakedpolynucelotide operatively coding for the polypeptide into theinterstitial space of a tissue comprising the cell, whereby the nakedpolynucleotide is taken up into the interior of the cell and has animmunogenic or pharmacological effect on the vertebrate. Also providedis a method for introducing a polynucleotide into muscle cells in vivo,comprising the steps of providing a composition comprising a nakedpolynucleotide in a pharmaceutically acceptable carrier, and contactingthe composition with muscle tissue of a vertebrate in vivo, whereby thepolynucleotide is introduced into muscle cells of the tissue. Thepolynucleotide may be an antisense polynucleotide. Alternatively, thepolynucleotide may code for a therapeutic peptide that is expressed bythe muscle cells after the contacting step to provide therapy to thevertebrate. Similarly, it may code for an immunogenic peptide that isexpressed by the muscle cells after the contacting step and whichgenerates an immune response, thereby immunizing the vertebrate.

One particularly attractive aspect of the invention is a method forobtaining long term administration of a polypeptide to a vertebrate,comprising the step of introducing a naked DNA sequence operativelycoding for the polypeptide interstitially into tissue of the vertebrate,whereby cells of the tissue produce the polypeptide for at least onemonth or at least 3 months, more preferably at least 6 months. In thisembodiment of the invention, the cells producing the polypeptide arenonproliferating cells, such as muscle cells.

Another method according to the invention is a method for obtainingtransitory expression of a polypeptide in a vertebrate, comprising thestep of introducing a naked mRNA sequence operatively coding for thepolypeptide interstitially into tissue of the vertebrate, whereby cellsof the tissue produce the polypeptide for less than about 20 days,usually less than about 10 days, and often less than 3 or 5 days. Formany of the methods of the invention, administration into solid tissueis preferred.

One important aspect of the invention is a method for treatment ofmuscular dystrophy, comprising the steps of introducing a therapeuticamount of a composition comprising a polynucleotide operatively codingfor dystrophin in a pharmaceutically acceptable injectable carrier invivo into muscle tissue of an animal suffering from muscular dystrophy,whereby the polynucleotide is taken up into the cells and dystrophin isproduced in vivo. Preferably, the polynucleotide is a nakedpolynucleotide and the composition is introduced interstitially into themuscle tissue.

The present invention also includes pharmaceutical products for all ofthe uses contemplated in the methods described herein. For example,there is a pharmaceutical product, comprising naked polynucleotide,operatively coding for a biologically active polypeptide, inphysiologically acceptable administrable form, in a container, and anotice associated with the container in form prescribed by agovernmental agency regulating the manufacture, use, or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the polynucleotide for human or veterinary administration.Such notice, for example, may be the labeling approved by the U.S. Foodand Drug Administration for prescription drugs, or the approved productinsert.

In another embodiment, the invention provides a pharmaceutical product,comprising naked polynucleotide, operatively coding for a biologicallyactive peptide, in solution in a physiologically acceptable injectablecarrier and suitable for introduction interstitially into a tissue tocause cells of the tissue to express the polypeptide, a containerenclosing the solution, and a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of manufacture, use, or sale of the solution ofpolynucleotide for human or veterinary administration. The peptide maybe immunogenic and administration of the solution to a human may serveto vaccinate the human, or an animal. Similarly, the peptide may betherapeutic and administration of the solution to a vertebrate in needof therapy relating to the polypeptide will have a therapeutic effect.

Also provided by the present invention is a pharmaceutical product,comprising naked antisense polynucleotide, in solution in aphysiologically acceptable injectable carrier and suitable forintroduction interstitially into a tissue to cause cells of the tissueto take up the polynucleotide and provide a therapeutic effect, acontainer enclosing the solution, and a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of manufacture, use, or sale of the solutionof polynucleotide for human or veterinary administration.

One particularly important aspect of the invention relates to apharmaceutical product for treatment of muscular dystrophy, comprising asterile, pharmaceutically acceptable carrier, a pharmaceuticallyeffective amount of a naked polynucleotide operatively coding fordystrophin in the carrier, and a container enclosing the carrier and thepolynucleotide in sterile fashion. Preferably, the polynucleotide isDNA.

From yet another perspective, the invention includes a pharmaceuticalproduct for use in supplying a biologically active polypeptide to avertebrate, comprising a pharmaceutically effective amount of a nakedpolynucleotide operatively coding for the polypeptide, a containerenclosing the carrier and the polynucleotide in a sterile fashion, andmeans associated with the container for permitting transfer of thepolynucleotide from the container to the interstitial space of a tissue,whereby cells of the tissue can take up and express the polynucleotide.The means for permitting such transfer can include a conventional septumthat can be penetrated, e.g., by a needle. Alternatively, when thecontainer is a syringe, the means may be considered to comprise theplunger of the syringe or a needle attached to the syringe. Containersused in the present invention will usually have at least 1, preferablyat least 5 to 10, and more preferably at least 50 to 100 micrograms ofpolynucleotide, to provide one or more unit dosages. For manyapplications, the container will have at least 500 micrograms or 1milligram, and often will contain at least 50 or 100 milligrams ofpolynucleotide.

Another aspect of the invention provides a pharmaceutical product foruse in immunizing a vertebrate, comprising a pharmaceutically effectiveamount of a naked polynucleotide operatively coding for an immunogenicpolypeptide, a sealed container enclosing the polynucleotide in asterile fashion, and means associated with the container for permittingtransfer of the polynucelotide from the container to the interstitialspace of a tissue, whereby cells of the tissue can take up and expressthe polynucleotide.

Still another aspect of the present invention is the use of nakedpolynucleotide operatively coding for a physiologically activepolypeptide in the preparation of a pharmaceutical for introductioninterstitially into tissue to cause cells comprising the tissue toproduce the polypeptide. The pharmaceutical, for example, may be forintroduction into muscle tissue whereby muscle cells produce thepolypeptide. Also contemplated is such use, wherein the peptide isdystrophin and the pharmaceutical is for treatment of musculardystrophy.

Another use according to the invention is use of naked antisensepolynucleotide in the preparation of a pharmaceutical for introductioninterstitially into tissue of a vertebrate to inhibit translation ofpolynucleotide in cells of the vertebrate.

The tissue into which the polynucleotide is introduced can be apersistent, non-dividing cell. The polynucleotide may be either a DNA orRNA sequence. When the polynucleotide is DNA, it can also be a DNAsequence which is itself non-replicating, but is inserted into aplasmid, and the plasmid further comprises a replicator. The DNA may bea sequence engineered so as not to integrate into the host cell genome.The polynucleotide sequences may code for a polypeptide which is eithercontained within the cells or secreted therefrom, or may comprise asequence which directs the secretion of the peptide.

The DNA sequence may also include a promoter sequence. In one preferredembodiment, the DNA sequence includes a cell-specific promoter thatpermits substantial transcription of the DNA only in predeterminedcells. The DNA may also code for a polymerase for transcribing the DNA,and may comprise recognition sites for the polymerase and the injectablepreparation may include an initial quantity of the polymerase.

In many instances, it is preferred that the polynucleotide is translatedfor a limited period of time so that the polypeptide delivery istransitory. The polypeptide may advantageously be a therapeuticpolypeptide, and may comprise an enzyme, a hormone, a lymphokine, areceptor, particularly a cell surface receptor, a regulatory protein,such as a growth factor or other regulatory agent, or any other proteinor peptide that one desires to deliver to a cell in a living vertebrateand for which corresponding DNA or mRNA can be obtained.

In preferred embodiments, the polynucleotide is introduced into muscletissue; in other embodiments the polynucleotide is incorporated intotissues of skin, brain, lung, liver, spleen or blood. The preparation isinjected into the vertebrate by a variety of routes, which may beintradermally, subdermally, intrathecally, or intravenously, or it maybe placed within cavities of the body. In a preferred embodiment, thepolynucleotide is injected intramuscularly. In still other embodiments,the preparation comprising the polynucleotide is impressed into theskin. Transdermal administration is also contemplated, as is inhalation.

In one preferred embodiment, the polynucleotide is DNA coding for both apolypeptide and a polymerase for transcribing the DNA, and the DNAincludes recognition sites for the polymerase and the injectablepreparation further includes a means for providing an initial quantityof the polymerase in the cell. The initial quantity of polymerase may bephysically present together with the DNA. Alternatively, it may beprovided by including mRNA coding therefor, which mRNA is translated bythe cell. In this embodiment of the invention, the DNA is preferably aplasmid. Preferably, the polymerase is phage T7 polymerase and therecognition site is a T7 origin of replication sequence.

In accordance with another aspect of the invention, there is provided amethod for treating a disease associated with the deficiency or absenceof a specific polypeptide in a vertebrate, comprising the steps ofobtaining an injectable preparation comprising a pharmaceuticallyacceptable injectable carrier containing a naked polynucleotide codingfor the specific polypeptide; introducing the injectable preparationinto a vertebrate and permitting the polynucleotide to be incorporatedinto a cell, wherein the polypeptide is formed as the translationproduct of the polynucleotide, and whereby the deficiency or absence ofthe polypeptide is compensated for. In preferred embodiments, thepreparation is introduced into muscle tissue and the method is appliedrepetitively. The method is advantageously applied where the deficiencyor absence is due to a genetic defect. The polynucleotide is preferablya non-replicating DNA sequence; the DNA sequence may also beincorporated into a plasmid vector which comprises an origin ofreplication.

In one of the preferred embodiments, the polynucleotide codes for anon-secreted polypeptide, and the polypeptide remains in situ. Accordingto this embodiment, when the polynucleotide codes for the polypeptidedystrophin, the method provides a therapy for Duchenne's syndrome;alternatively, when the polynucleotide codes for the polypeptidephenylalanine hydroxylase, the method comprises a therapy forphenylketonuria. In another preferred embodiment of the method, thepolynucleotide codes for a polypeptide which is secreted by the cell andreleased into the circulation of the vertebrate; in a particularlypreferred embodiment the polynucleotide codes for human growth hormone.

In yet another embodiment of the method, there is provided a therapy forhypercholesterolemia wherein a polynucleotide coding for a receptorassociated with cholesterol homeostasis is introduced into a liver cell,and the receptor is expressed by the cell.

In accordance with another aspect of the present invention, there isprovided a method for immunizing a vertebrate, comprising the steps ofobtaining a preparation comprising an expressible polynucleotide codingfor an immunogenic translation product, and introducing the preparationinto a vertebrate wherein the translation product of the polynucleotideis formed by a cell of the vertebrate, which elicits an immune responseagainst the immunogen. In one embodiment of the method, the injectablepreparation comprises a pharmaceutically acceptable carrier containingan expressible polynucleotide coding for an immunogenic peptide, and onthe introduction of the preparation into the vertebrate, thepolynucleotide is incorporated into a cell of the vertebrate wherein animmunogenic translation product of the polynucleotide is formed, whichelicits an immune response against the immunogen.

In an alternative embodiment, the preparation comprises one or morecells obtained from the vertebrate and transfected in vitro with thepolynucleotide, whereby the polynucleotide is incorporated into saidcells, where an immunogenic translation product of the polynucleotide isformed, and whereby on the introduction of the preparation into thevertebrate, an immune response against the immunogen is elicited. In anyof the embodiments of the invention, the immunogenic product may besecreted by the cells, or it may be presented by a cell of thevertebrate in the context of the major histocompatibility antigens,thereby eliciting an immune response against the immunogen. The methodmay be practiced using non-dividing, differentiated cells from thevertebrates, which cells may be lymphocytes, obtained from a bloodsample; alternatively, it may be practiced using partiallydifferentiated skin fibroblasts which are capable of dividing. In apreferred embodiment, the method is practiced by incorporating thepolynucleotide coding for an immunogenic translation product into muscletissue.

The polynucleotide used for immunization is preferably an mRNA sequence,although a non-replicating DNA sequence may be used. The polynucleotidemay be introduced into tissues of the body using the injectable carrieralone; liposomal preparations are preferred for methods in which invitro transfections of cells obtained from the vertebrate are carriedout.

The carrier preferably is isotonic, hypotonic, or weakly hypertonic, andhas a relatively low ionic strength, such as provided by a sucrosesolution. The preparation may further advantageously comprise a sourceof a cytokine which is incorporated into lipsomes in the form of apolypeptide or as a polynucleotide.

The method may be used to selectively elicit a humoral immune response,a cellular immune response, or a mixture of these. In embodimentswherein the cell expresses major histocompatibility complex of Class I,and the immunogenic peptide is presented in the context of the Class Icomplex, the immune response is cellular and comprises the production ofcytotoxic T-cells.

In one such embodiment, the immunogenic peptide is associated with avirus, is presented in the context of Class I antigens, and stimulatescytotoxic T-cells which are capable of destroying cells infected withthe virus. A cytotoxic T-cell response may also be produced accordingthe method where the polynucleotide codes for a truncated viral antigenlacking humoral epitopes.

In another of these embodiments, the immunogenic peptide is associatedwith a tumor, is presented in the context of Class I antigens, andstimulates cytotoxic T cells which are capable of destroying tumorcells. In yet another embodiment wherein the injectable preparationcomprises cells taken from the animal and transfected in vitro, thecells expressing major histocompatibility antigen of class I and classII, and the immune response is both humoral and cellular and comprisesthe production of both antibody and cytotoxic T-cells.

In another embodiment, there is provided a method of immunizing avertebrate, comprising the steps of obtaining a positively chargedliposome containing an expressible polynucleotide coding for animmunogenic peptide, and introducing the liposome into a vertebrate,whereby the liposome is incorporated into a monocyte, a macrophage, oranother cell, where an immunogenic translation product of thepolynucleotide is formed, and the product is processed and presented bythe cell in the context of the major histocompatibility complex, therebyeliciting an immune response against the immunogen. Again, thepolynucleotide is preferably mRNA, although DNA may also be used. And asbefore, the method may be practiced without the liposome, utilizing justthe polynucleotide in an injectable carrier.

The present invention also encompasses the use of DNA coding for apolypeptide and for a polymerase for transcribing the DNA, and whereinthe DNA includes recognition sites for the polymerase. The initialquantity of polymerase is provided by including mRNA coding therefor inthe preparation, which mRNA is translated by the cell. The mRNApreferably is provided with means for retarding its degradation in thecell. This can include capping the mRNA, circularizing the mRNA, orchemically blocking the 5′ end of the mRNA. The DNA used in theinvention may be in the form of linear DNA or may be a plasmid. EpisomalDNA is also contemplated. One preferred polymerase is phage T7 RNApolymerase and a preferred recognition site is a T7 RNA polymerasepromoter.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention requires obtaining nakedpolynucleotide operatively coding for a polypeptide for incorporationinto vertebrate cells. A polynucleotide operatively codes for apolypeptide when it has all the genetic information necessary forexpression by a target cell, such as promoters and the like. Thesepolynucleotides can be administered to the vertebrate by any method thatdelivers injectable materials to cells of the vertebrate, such as byinjection into the interstitial space of tissues such as muscles orskin, introduction into the circulation or into body cavities or byinhalation or insufflation. A naked polynucleotide is injected orotherwise delivered to the animal with a pharmaceutically acceptableliquid carrier. In preferred applications, the liquid carrier is aqueousor partly aqueous, comprising sterile, pyrogen-free water. The pH of thepreparation is suitably adjusted and buffered. The polynucleotide cancomprise a complete gene, a fragment of a gene, or several genes,together with recognition and other sequences necessary for expression.

In the embodiments of the invention that require use of liposomes, forexample, when the polynucleotide is to be associated with a lipsome, itrequires a material for forming liposomes, preferably cationic orpositively charged liposomes, and requires that liposomal preparationsbe made from these materials. With the liposomal material in hand, thepolynucleotide may advantageously be used to transfect cells in vitrofor use as immunizing agents, or to administer polynucleotides intobodily sites where liposomes may be taken up by phagocytic cells.

Polynucleotide Materials

The naked polynucleotide materials used according to the methods of theinvention comprise DNA and RNA sequences or DNA and RNA sequences codingfor polypeptides that have useful therapeutic applications. Thesepolynucleotide sequences are naked in the sense that they are free fromany delivery vehicle that can act to facilitate entry into the cell, forexample, the polynucleotide sequences are free of viral sequences,particularly any viral particles which may carry genetic information.They are similarly free from, or naked with respect to, any materialwhich promotes transaction, such as liposomal formulations, chargedlipids such as Lipofectin™ or precipitating agents such as CaPO₄.

The DNA sequences used in these methods can be those sequences which donot integrate into the genome of the host cell. These may benon-replicating DNA sequences, or specific replicating sequencesgenetically engineered to lack the genome-integration ability.

The polynucleotide sequences of the invention are DNA or RNA sequenceshaving a therapeutic effect after being taken up by a cell. Examples ofpolynucleotides that are themselves therapeutic are anti-sense DNA andRNA; DNA coding for an anti-sense RNA; or DNA coding for tRNA or rRNA toreplace defective or deficient endogenous molecules. The polynucleotidesof the invention can also code for therapeutic polypeptides. Apolypeptide is understood to be any translation product of apolynucleotide regardless of size, and whether glycosylated or not.Therapeutic polypeptides include as a primary example, thosepolypeptides that can compensate for defective or deficient species inan animal, or those that act through toxic effects to limit or removeharmful cells from the body.

Therapeutic polynucleotides provided by the invention can also code forimmunity-conferring polypeptides, which can act as endogenous immunogensto provoke a humoral or cellular response, or both. The polynucleotidesemployed according to the present invention can also code for anantibody. In this regard, the term “antibody” encompasses wholeimmunoglobulin of any class, chimeric antibodies and hybrid antibodieswith dual or multiple antigen or epitope specificities, and fragments,such as F(ab)₂, Fab′, Fab and the like, including hybrid fragments. Alsoincluded within the meaning of “antibody” are conjugates of suchfragments, and so-called antigen binding proteins (single chainantibodies) as described, for example, in U.S. Pat. No. 4,704,692, thecontents of which are hereby incorporated by reference.

Thus, an isolated polynucleotide coding for variable regions of anantibody can be introduced, in accordance with the present invention, toenable the treated subject to produce antibody in situ. For illustrativemethodology, relating to obtaining antibody-encoding polynucleotides,see Ward et al. Nature, 341:544-546 (1989); Gillies et al., Biotechnol.7:799-804 (1989); and Nakatani et al., loc. cit., 805-810 (1989). Theantibody in turn would exert a therapeutic effect, for example, bybinding a surface antigen associated with a pathogen. Alternatively, theencoded antibodies can be anti-idiotypic antibodies (antibodies thatbind other antibodies) as described, for example, in U.S. Pat. No.4,699,880. Such anti-idiotypic antibodies could bind endogenous orforeign antibodies in a treated individual, thereby to ameliorate orprevent pathological conditions associated with an immune response,e.g., in the context of an autoimmune disease.

Polynucleotide sequences of the invention preferably code fortherapeutic or immunogenic polypeptides, and these sequences may be usedin association with other polynucleotide sequences coding for regulatoryproteins that control the expression of these polypeptides. Theregulatory protein can act by binding to genomic DNA so as to regulateits transcription; alternatively, it can act by binding to messenger RNAto increase or decrease its stability or translation efficiency.

The polynucleotide material delivered to the cells in vivo can take anynumber of forms, and the present invention is not limited to anyparticular polynucleotide coding for any particular polypeptide.Plasmids containing genes coding for a large number of physiologicallyactive peptides and antigens or immunogens have been reported in theliterature and can be readily obtained by those of skill in the art.

Where the polynucleotide is to be DNA, promoters suitable for use invarious vertebrate systems are well known. For example, for use inmurine systems, suitable strong promoters include RSV LTR, MPSV LTR,SV40 IEP, and metallothionein promoter. In humans, on the other hand,promoters such as CMV IEP may advantageously be used. All forms of DNA,whether replicating or non-replicating, which do not become integratedinto the genome, and which are expressible, are within the methodscontemplated by the invention.

With the availability of automated nucleic acid synthesis equipment,both DNA and RNA can be synthesized directly when the nucleotidesequence is known or by a combination of PCR cloning and fermentation.Moreover, when the sequence of the desired polypeptide is known, asuitable coding sequence for the polynucleotide can be inferred.

When the polynucleotide is mRNA, it can be readily prepared from thecorresponding DNA in vitro. For example, conventional techniques utilizephage RNA polymerases SP6, T3, or T7 to prepare mRNA from DNA templatesin the presence of the individual ribonucleoside triphosphates. Anappropriate phage promoter, such as a T7 origin of replication site isplaced in the template DNA immediately upstream of the gene to betranscribed. Systems utilizing T7 in this manner are well known, and aredescribed in the literature, e.g., in Current Protocols in MolecularBiology, §3.8 (Vol. 1 1988).

One particularly preferred method for obtaining the mRNA used in thepresent invention is set forth in Examples 2-5. In general, however, itshould be apparent that the pXGB plasmid or any similar plasmid that canbe readily constructed by those of ordinary skill in the art can be usedwith a virtually unlimited number of cDNAs in practicing the presentinvention. Such plasmids may advantageously comprise a promoter for adesired RNA polymerase, followed by a 5′ untranslated region, a 3′untranslated region, and a template for a poly A tract. There should bea unique restriction site between these 5′ and 3′ regions to facilitatethe insertion of any desired cDNA into the plasmid. Then, after cloningthe plasmid containing the desired gene, the plasmid is linearized bycutting in the polyadenylation region and is transcribed in vitro toform mRNA transcripts. These transcripts are preferably provided with a5′ cap, as demonstrated in Example 5. Alternatively, a 5′ untranslatedsequence such as EMC can be used which does not require a 5′ cap.

While the foregoing represents a preferred method for preparing themRNA, it will be apparent to those of skill in the art that manyalternative methods also exist. For example, the mRNA can be prepared incommercially-available nucleotide synthesis apparatus. Alternatively,mRNA in circular form can be prepared. Exonuclease-resistant RNAs suchas circular mRNA, chemically blocked mRNA, and mRNA with a 5′ cap arepreferred, because of their greater half-life in vivo.

In particular, one preferred mRNA is a self-circularizing mRNA havingthe gene of interest preceded by the 5′ untranslated region of poliovirus. It has been demonstrated that circular mRNA has an extremely longhalf-life (Harland & Misher, Development 102: 837-852 (1988)) and thatthe polio virus 5′ untranslated region can promote translation of mRNAwithout the usual 5′ cap (Pelletier & Sonnenberg, Nature 334:320-325(1988), hereby incorporated by reference).

This material may be prepared from a DNA template that is self-splicingand generates circular “lariat” mRNAs, using the method of Been & Cech,Cell 47:206-216 (1986) (hereby incorporated by reference). We modifythat template by including the 5′ untranslated region of the polio virusimmediately upstream of the gene of interest, following the procedure ofManiatis, T. et al. MOLECULAR CLONING: A LABORATORY MANUAL, Cold SpringHarbor, N.Y. (1982).

In addition, the present invention includes the use of mRNA that ischemically blocked at the 5′ and/or 3′ end to prevent access by RNAse.(This enzyme is an exonuclease and therefore does not cleave RNA in themiddle of the chain.) Such chemical blockage can substantially lengthenthe half life of the RNA in vivo. Two agents which may be used to modifyRNA are available from Clonetech Laboratories, Inc., Palo Alto, Calif.:C2 AminoModifier (Catalog §5204-1) and Amino-7-dUTP (Catalog § K1022-1).These materials add reactive groups to the RNA. After introduction ofeither of these agents onto an RNA molecule of interest, an appropriatereactive substituent can be linked to the RNA according to themanufacturer's instructions. By adding a group with sufficient bulk,access to the chemically modified RNA by RNAse can be prevented.

Transient Gene Therapy

Unlike gene therapies proposed in the past, one major advantage of thepresent invention is the transitory nature of the polynucleotidesynthesis in the cells. (We refer to this as reversible gene therapy, orTGT.) With mRNA introduced according to the present invention, theeffect will generally last about one day. Also, in marked contrast togene therapies proposed in the past, mRNA does not have to penetrate thenucleus to direct protein synthesis; therefore, it should have nogenetic liability.

In some situations, however, a more prolonged effect may be desiredwithout incorporation of the exogenous polynucleic acid into the genomeof the host organism. In order to provide such an effect, a preferredembodiment of the invention provides introducing a DNA sequence codingfor a specific polypeptide into the cell. We have found, according tothe methods of the invention, that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of about up to six months, and we have observed no evidenceof integration of the DNA sequences into the genome of the cells.Alternatively, an even more prolonged effect can be achieved byintroducing the DNA sequence into the cell by means of a vector plasmidhaving the DNA sequence inserted therein. Preferably, the plasmidfurther comprises a replicator. Such plasmids are well known to thoseskilled in the art, for example, plasmid pBR322, with replicator pMB1,or plasmid pMK16, with replicator ColE1 (Ausubel, Current Portocols inMolecular Biology, John Wiley and Sons, New York (1988) §II:1.5.2.

Results of studies of the time course of expression of DNA and mRNAintroduced into muscle cells as described in Examples 1 and 13 indicatethat mRNA expression is more rapid, although shorter in duration thanDNA expression. An immediate and long lived gene expression can beachieved by administering to the cell a liposomal preparation comprisingboth DNA and an RNA polymerase, such as the phage polymerases T7, T3,and SP6. The liposome also includes an initial source of the appropriateRNA polymerase, by either including the actual enzyme itself, oralternatively, an mRNA coding for that enzyme. When the liposome isintroduced into the organism, it delivers the DNA and the initial sourceof RNA polymerase to the cell. The RNA polymerase, recognizing thepromoters on the introduced DNA, transcribes both genes, resulting intranslation products comprising more RNA polymerase and the desiredpolypeptide. Production of these materials continues until theintroduced DNA (which is usually in the form of a plasmid) is degraded.In this manner, production of the desired polypeptide in vivo can beachieved in a few hours and be extended for one month or more.

Although not limited to the treatment of genetic disease, the methods ofthe invention can accordingly be appropriately applied to treatmentstrategies requiring delivery and functional expression of missing ordefective genes.

The polynucleotides may be delivered to the interstitial space oftissues of the animal body, including those of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. We have discovered that in vivo muscle cells areparticularly competent in their ability to take up and expresspolynucleotides. This ability may be due to the singular tissuearchitecture of muscle, comprising multinucleated cells, sarcoplasmicreticulum, and transverse tubular system. Polynucleotides may enter themuscle through the transverse tubular system, which containsextracellular fluid and extends deep into the muscle cell. It is alsopossible that the polynucleotides enter damaged muscle cells which thenrecover.

Muscle is also advantageously used as a site for the delivery andexpression of polynucleotides in a number of therapeutic applicationsbecause animals have a proportionately large muscle mass which isconveniently accessed by direct injection through the skin; for thisreason, a comparatively large dose of polynucleotides can be depositedin muscle by multiple injections, and repetitive injections, to extendtherapy over long periods of time, are easily performed and can becarried out safely and without special skill or devices.

Muscle tissue can be used as a site for injection and expression ofpolynucleotides in a set of general strategies, which are exemplary andnot exhaustive. First, muscle disorders related to defective or absentgene products can be treated by introducing polynucleotides coding for anon-secreted gene product into the diseased muscle tissue. In a secondstrategy, disorders of other organs or tissues due to the absence of agene product, and which results in the build-up of a circulating toxicmetabolite can be treated by introducing the specific therapeuticpolypeptide into muscle tissue where the non-secreted gene product isexpressed and clears the circulating metabolite. In a third strategy, apolynucleotide coding for an secretable therapeutic polypeptide can beinjected into muscle tissue from where the polypeptide is released intothe circulation to seek a metabolic target. This use is demonstrated inthe expression of growth hormone gene injected into muscle, Example 18.Certain DNA segments, are known to serve as “signals” to directsecretion (Wickner, W. T. and H. F. Lodish, Science 230:400-407 (1985),and these may be advantageously employed. Finally, in immunizationstrategies, muscle cells may be injected with polynucleotides coding forimmunogenic peptides, and these peptides will be presented by musclecells in the context of antigens of the major histocompatibility complexto provide a selected immune response against the immunogen.

Tissues other than those of muscle, and having a less efficient uptakeand expression of injected polynucleotides, may nontheless beadvantageously used as injection sites to produce therapeuticpolypeptides or polynucleotides under certain conditions. One suchcondition is the use of a polynucleotide to provide a polypeptide whichto be effective must be present in association with cells of a specifictype; for example, the cell surface receptors of liver cells associatedwith cholesterol homeostasis. (Brown and Goldstein, Science 232:34-37(1986)). In this application, and in many others, such as those in whichan enzyme or hormone is the gene product, it is not necessary to achievehigh levels of expression in order to effect a valuable therapeuticresult.

One application of TGT is in the treatment of muscular dystrophy. Thegenetic basis of the muscular dystrophies is just beginning to beunraveled. The gene related to Duchenne/Becker muscular dystrophy hasrecently been cloned and encodes a rather large protein, termeddystrophin. Retroviral vectors are unlikely to be useful, because theycould not accommodate the rather large size of the cDNA (about 13 kb)for dystrophin. Very recently reported work is centered on transplantingmyoblasts, but the utility of this approach remains to be determined.Clearly, an attractive approach would be to directly express thedystrophin gene within the muscle of patients with Duchennes. Since mostpatients die from respiratory failure, the muscles involved withrespiration would be a primary target.

Another application is in the treatment of cystic fibrosis. The gene forcystic fibrosis was recently identified (Goodfellow, P. Nature, 341(6238):102-3 (Sep. 14, 1989); Rommens, J. et al. Science,245(4922):1059-1065 (Sep. 8, 1989); Beardsley, T. et al., ScientificAmerican, 261(5):28-30 (1989). Significant amelioration of the symptomsshould be attainable by the expression of the dysfunctional proteinwithin the appropriate lung cells. The bronchial epithelial cells arepostulated to be appropriate target lung cells and they could beaccessible to gene transfer following instillation of genes into thelung. Since cystic fibrosis is an autosomal recessive disorder one wouldneed to achieve only about 5% of normal levels of the cystic fibrosisgene product in order to significantly ameliorate the pulmonarysymptoms.

Biochemical genetic defects of intermediary metabolism can also betreated by TGT. These diseases include phenylketonuria, galactosemia,maple-syrup urine disease, homocystinuria, propionic acidemia,methylmalonic acidemia, and adenosine deaminase deficiency. Thepathogenesis of disease in most of these disorders fits thephenylketonuria (PKU) model of a circulating toxic metabolite. That is,because of an enzyme block, a biochemical, toxic to the body,accumulates in body fluids. These disorders are ideal for gene therapyfor a number of reasons. First, only 5% of normal levels of enzymeactivity would have to be attained in order to significantly clearenough of the circulating toxic metabolite so that the patient issignificantly improved. Second, the transferred gene could most often beexpressed in a variety of tissues and still be able to clear the toxicbiochemical.

Reversible gene therapy can also be used in treatment strategiesrequiring intracytoplasmic or intranuclear protein expression. Someproteins are known that are capable of regulating transcription bybinding to specific promoter regions on nuclear DNA. Other proteins bindto RNA, regulating its degradation, transport from the nucleus, ortranslation efficiency. Proteins of this class must be deliveredintracellularly for activity. Extracellular delivery of recombinanttranscriptional or translational regulatory proteins would not beexpected to have biological activity, but functional delivery of the DNAor RNA by TGT would be active. Representative proteins of this type thatwould benefit from TGT would include NEF, TAT, steroid receptor and theretinoid receptor.

Gene therapy can be used in a strategy to increase the resistance of anAIDS patient to HIV infection. Introducing an AIDS resistance gene, suchas, for example, the NEF gene or the soluble CD4 gene to preventbudding, into an AIDS patient's T cells will render his T cells lesscapable of producing active AIDS virus, thus sparing the cells of theimmune system and improving his ability to mount a T cell dependentimmune response. Thus, in accordance with the invention, a population ofthe AIDS patient's own T cells is isolated from the patient's blood.These cells are then transfected in vitro and then reintroduced backinto the patient's blood. The virus-resistant cells will have aselective advantage over the normal cells, and eventually repopulate thepatient's lymphatic system. DNA systemic delivery to macrophages orother target cells can be used in addition to the extracorporealtreatment strategy. Although this strategy would not be expected toeradicate virus in the macrophage reservoir, it will increase the levelof T cells and improve the patient's immune response.

In all of the systemic strategies presented herein, an effective DNA ormRNA dosage will generally be in the range of from about 0.05 μg/kg toabout 50 mg/kg, usually about 0.005-5 mg/kg. However, as will beappreciated, this dosage will vary in a manner apparent to those ofskill in the art according to the activity of the peptide coded for bythe DNA or mRNA and the particular peptide used. For delivery ofadenosine deaminase to mice or humans, for example, adequate levels oftranslation are achieved with a DNA or mRNA dosage of about 0.5 to 5mg/kg. See Example 10. From this information, dosages for other peptidesof known activity can be readily determined.

Diseases which result from deficiencies of critical proteins may beappropriately treated by introducing into specialized cells, DNA or mRNAcoding for these proteins. A variety of growth factors such as nervegrowth factor and fibroblast growth factor have been shown to affectneuronal cell survival in animal models of Alzheimer's disease. In theaged rat model, NGF infusions have reversed the loss of cholinergicneurons. In the fimbria-fornix lesion rat, NGF infusions or secretionfrom genetically-modified fibroblasts have also avoided the loss ofcholinergic function. Cholinergic activity is diminished in patientswith Alzheimer's. The expression within the brain of transduced genesexpressing growth factors could reverse the lost of function of specificneuronal groups.

Introduction of DNA or mRNA by transfection of the gene for neuronalgrowth factor into cells lining the cranial cavity can be used inaccordance with the present invention in the treatment of Alzheimer'sdisease. In particular, the present invention treats this disease byintracranial injection of from about 10 μg to about 100 μg of DNA ormRNA into the parenchyma through use of a stereotaxic apparatus.Specifically, the injection is targeted to the cholinergic neurons inthe medial septum. The DNA or mRNA injection is repeated every 1-3 daysfor 5′ capped, 3′ polyadenylated mRNA, and every week to 21 days forcircular mRNA, and every 30 to 60 days for DNA. Injection of DNA inaccordance with the present invention is also contemplated. DNA would beinjected in corresponding amounts; however, frequency is injection wouldbe greatly reduced. Episomal DNA, for example, could be active for anumber of months, and reinjection would only be necessary upon notableregression by the patient.

In addition, the enzymes responsible for neurotransmitter synthesiscould be expressed from transduced genes. For example, the gene forcholine acetyl transferase could be expressed within the brain cells(neurons or glial) of specific areas to increase acetylcholine levelsand improve brain function.

The critical enzymes involved in the synthesis of otherneurotransmitters such as dopamine, norepinephrine, and GABA have beencloned and available. The critical enzymes could be locally increased bygene transfer into a localized area of the brain. The increasedproductions of these and other neurotransmitters would have broadrelevance to manipulation of localized neurotransmitter function andthus to a broad range of brain disease in which disturbedneurotransmitter function plays a crucial role. Specifically, thesediseases could include schizophrenia and manic-depressive illnesses andParkinson's Disease. It is well established that patients withParkinson's suffer from progressively disabled motor control due to thelack of dopamine synthesis within the basal ganglia. The rate limitingstep for dopamine synthesis is the conversion of tyrosine to L-DOPA bythe enzyme, tyrosine hydroxylase. L-DOPA is then converted to dopamineby the ubiquitous enzyme, DOPA decarboxylase. That is why thewell-established therapy with L-DOPA is effective (at least for thefirst few years of treatment). Gene therapy could accomplish the similarpharmacologic objective by expressing the genes for tyrosine hydroxylaseand possible DOPA decarboxylase as well. Tyrosine is readily availablewithin the CNS.

The genetic form of alpha-1-antitrypsin deficiency can result in bothliver and lung disease. The liver disease, which is less common, iscaused by the accumulation of an abnormal protein and would be lessamenable to gene therapy. The pulmonary complications, however, would beamenable to the increased expression of alpha-1-antitrypsin within thelung. This should prevent the disabling and eventually lethal emphysemafrom developing.

Alpha-1-antitrypsin deficiency also occurs in tobacco smokers sincetobacco smoke decreases alpha-1-antitrypsin activity and thus serineprotease activity that leads to emphysema. In addition, some recent datalinks tobacco smoke's anti-trypsin effect to aneurysms of the aorta.Aneurysms would also be preventable by raising blood levels. ofanti-1-antitrypsin since this would decrease protease activity thatleads to aneurysms.

Patients with degenerative disease of the lung could also benefit fromthe expression of enzymes capable of removing other toxic metaboliteswhich tend to accumulate in diseased lung tissue. Superoxide dismutaseand catalase could be delivered by TGT to ameliorate these problems.

TGT can be used in treatment strategies requiring the delivery of cellsurface receptors. It could be argued that there is no need to deciphermethodology for functional in vivo delivery of genes. There is, afterall, an established technology for the synthesis and large scaleproduction of proteins, and proteins are then end product of geneexpression. This logic applies for many protein molecules which actextracellularly or interact with cell surface receptors, such as tissueplasminogen activator (TPA), growth hormone, insulin, interferon,granulocyte-macrophage colony stimulating factor (GMCSF), erythropoietin(EPO), etc. However, the drug delivery problems associated with properlydelivering a recombinant cell surface receptor to be inserted in theplasma membrane of its target cell in the proper orientation for afunctional receptor have hithertofore appeared intractable.

When DNA or RNA coding for a cell surface receptor is deliveredintracellularly in accordance with the present invention, the resultingprotein can be efficiently and functionally expressed on the target cellsurface. If the problem of functional delivery of recombinant cellsurface receptors remains intractable, them the only way of approachingthis therapeutic modality will be through gene delivery. Similar logicfor nuclear or cytoplasmic regulation of gene expression applies tonuclear regulatory factor bound to DNA to regulate (up or down) RNAtranscription and to cytoplasmic regulatory factors which bind to RNA toincrease or decrease translational efficiency and degradation. TGT couldin this way provide therapeutic strategies for the treatment of cysticfibrosis, muscular dystrophy and hypercholesterolemia.

Elevated levels of cholesterol in the blood may be reduced in accordancewith the present invention by supplying mRNA coding for the LDL surfacereceptor to hepatocytes. A slight elevation in the production of thisreceptor in the liver of patients with elevated LDL will havesignificant therapeutic benefits. Therapies based on systemicadministration of recombinant proteins are not able to compete with thepresent invention, because simply administering the recombinant proteincould not get the receptor into the plasma membrane of the target cells.The receptor must be properly inserted into the membrane in order toexert its biological effect. It is not usually necessary to regulate thelevel of receptor expression; the more expression the better. Thissimplifies the molecular biology involved in preparation of the mRNA foruse in the present invention. For example, lipid/DNA or RNA complexescontaining the LDL receptor gene may be prepared and supplied to thepatient by repetitive I.V. injections. The lipid complexes will be takenup largely by the liver. Some of the complexes will be taken up byhepatocytes. The level of LDL receptor in the liver will increasegradually as the number of injections increases. Higher liver LDLreceptor levels will lead to therapeutic lowering of LDL andcholesterol. An effective mRNA dose will generally be from about 0.1 toabout 5 mg/kg.

Other examples of beneficial applications of TGT include theintroduction of the thymidine kinase gene into macrophages of patientsinfected with the HIV virus. Introduction of the thymidine kinase geneinto the macrophage reservoir will render those cells more capable ofphosphorylating AZT. This tends to overcome their resistance to AZTtherapy, making AZT capable of eradicating the HIV reservoir inmacrophages. Lipid/DNA complexes containing the thymidine kinase genecan be prepared and administered to the patient through repetitiveintravenous injections. The lipid complexes will be taken up largely bythe macrophage reservoir leading to elevated levels of thymidine kinasein the macrophages. This will render the AZT resistant cells subject totreatment with AZT. The thymidine kinase therapy can also be focused byputting the thymidine kinase gene under the control of the HTLV IIIpromoter. According to this strategy, the thymidine kinase would only besynthesized on infection of the cell by HIV virus, and the production ofthe tat protein which activates the promoter. An analogous therapy wouldsupply cells with the gene for diphtheria toxin under the control of thesame HTLV III promoter, with the lethal result occurring in the cellsonly after HIV infection.

These AIDS patients could also be treated by supplying the interferongene to the macrophages according to the TGT method. Increased levels oflocalized interferon production in macrophages could render them moreresistant to the consequences of HIV infection. While local levels ofinterferon would be high, the overall systemic levels would remain low,thereby avoiding the systemic toxic effects like those observed afterrecombinant interferon administration. Lipid/DNA or RNA complexescontaining the interferon gene can be prepared and administered to thepatient by repetitive intravenous injections. The lipid complexes willbe taken up largely by the macrophage reservoir leading to elevatedlocalized levels of interferon in the macrophages. This will render themless susceptible to HIV infection.

Various cancers may be treated using TGT by supplying a diphtheria toxingene on a DNA template with a tissue specific enhancer to focusexpression of the gene in the cancer cells. Intracellular expression ofdiphtheria toxin kills cells. These promoters could be tissue-specificsuch as using a pancreas-specific promoter for the pancreatic cancer. Afunctional diphtheria toxin gene delivered to pancreatic cells coulderadicate the entire pancreas. This strategy could be used as atreatment for pancreatic cancer. The patients would have noinsurmountable difficulty surviving without a pancreas. The tissuespecific enhancer would ensure that expression of diphtheria toxin wouldonly occur in pancreatic cells. DNA/lipid complexes containing thediphtheria toxin gene under the control of a tissue specific enhancerwould be introduced directly into a cannulated artery feeding thepancreas. The infusion would occur on some dosing schedule for as longas necessary to eradicate the pancreatic tissue. Other lethal genesbesides diphtheria toxin could be used with similar effect, such asgenes for ricin or cobra venom factor or enterotoxin.

Also, one could treat cancer by using a cell-cycle specific promoterthat would only kill cells that are rapidly cycling (dividing) such ascancer cells. Cell-cycle specific filling could also be accomplished bydesigning mRNA encoding killer proteins that are stable only in cyclingcells (i.e. histone mRNA that is only stable during S phase). Also, onecould use developmental-specific promoters such as the use ofalpha-fetoprotein that is only expressed in fetal liver cells and inhepatoblastoma cells that have dedifferentiated into a more fetal state.

One could also treat specialized cancers by the transfer of genes suchas the retinoblastoma gene (and others of that family) that suppress thecancer properties of certain cancers.

The TGT strategy can be used to provide a controlled, sustained deliveryof peptides. Conventional drugs, as well as recombinant protein drugs,can benefit from controlled release devices. The purpose of thecontrolled release device is to deliver drugs over a longer time period,so that the number of doses required is reduced. This results inimprovements in patient convenience and compliance. There are a widevariety of emerging technologies that are intended to achieve controlledrelease.

TGT can be used to obtain controlled delivery of therapeutic peptides.Regulated expression can be obtained by using suitable promoters,including cell-specific promoters. Suitable peptides delivered by thepresent invention include, for example, growth hormone, insulin,interleukins, interferons, GMCSF, EPO, and the like. Depending on thespecific application, the DNA or an RNA construct selected can bedesigned to result in a gene product that is secreted from the injectedcells and into the systemic circulation.

TGT can also comprise the controlled delivery of therapeuticpolypeptides or peptides which is achieved by including with thepolynucleotide to be expressed in the cell, an additional polynucleotidewhich codes for a regulatory protein which controls processes oftranscription and translation. These polynucleotides comprise thosewhich operate either to up regulate or down regulate polypeptideexpression, and exert their effects either within the nucleus or bycontrolling protein translation events in the cytoplasma.

The T7 polymerase gene can be used in conjunction with a gene ofinterest to obtain longer duration of effect of TGT. Episomal DNA suchas that obtained from the origin of replication region for the EpsteinBarr virus can be used, as well as that from other origins ofreplication which are functionally active in mammalian cells, andpreferably those that are active in human cells. This is a way to obtainexpression from cells after many cell divisions, without riskingunfavorable integration events that are common to retrovirus vectors.Controlled release of calcitonin could be obtained if a calcitonin geneunder the control of its own promoter could be functionally introducedinto some site, such as liver or skin. Cancer patients withhypercalcemia would be a group to whom this therapy could be applied.

Other gene therapies using TGT can include the use of a polynucleotidethat has a therapeutic effect without being translated into apolypeptide. For example, TGT can be used in the delivery of anti-sensepolynucleotides for turning off the expression of specific genes.Conventional anti-sense methodology suffers from poor efficacy, in part,because the oligonucleotide sequences delivered are too short. With TGT,however, full length anti-sense sequences can be delivered as easily asshort oligomers. Anti-sense polynucleotides can be DNA or RNA moleculesthat themselves hybridize to (and, thereby, prevent transcription ortranslation of) an endogenous nucleotide sequence. Alternatively, ananti-sense DNA may encode an RNA the hybridizes to an endogenoussequence, interfering with translation. Other uses of TGT in this veininclude delivering a polynucleotide that encodes a tRNA or rRNA toreplace a defective or deficient endogenous tRNA or rRNA, the presenceof which causes the pathological condition.

Cell-specific promoters can also be used to permit expression of thegene only in the target cell. For example, certain genes are highlypromoted in adults only in particular types of tumors. Similarly,tissue-specific promoters for specialized tissue, e.g., lens tissue ofthe eye, have also been identified and used in heterologous expressionsystems.

Beyond the therapies described, the method of the invention can be usedto deliver polynucleotides to animal stock to increase production ofmilk in dairy cattle or muscle mass in animals that are raised for meat.

DNA and mRNA Vaccines

According to the methods of the invention, both expressible DNA and mRNAcan be delivered to cells to form therein a polypeptide translationproduct. If the nucleic acids contain the proper control sequences, theywill direct the synthesis of relatively large amounts of the encodedprotein. When the DNA and mRNA delivered to the cells codes for animmunizing peptide, the methods can be applied to achieve improved andmore effective immunity against infectious agents, includingintracellular viruses, and also against tumor cells.

Since the immune systems of all vertebrates operate similarly, theapplications described can be implemented in all vertebrate systems,comprising mammalian and avian species, as well as fish.

The methods of the invention may be applied by direct injection of thepolynucleotide into cells of the animal in vivo, or by in vitrotransfection of some of the animal cells which are then re-introducedinto the animal body. The polynucleotides may be delivered to variouscells of the animal body, including muscle, skin, brain, lung, liver,spleen, or to the cells of the blood. Delivery of the polynucleotidesdirectly in vivo is preferably to the cells of muscle or skin. Thepolynucleotides may be injected into muscle or skin using an injectionsyringe. They may also be delivered into muscle or skin using a vaccinegun.

It has recently been shown that cationic lipids can be used tofacilitate the transfection of cells in certain applications,particularly in vitro transfection. Cationic lipid based transfectiontechnology is preferred over other methods; it is more efficient andconvenient than calcium phosphate, DEAE dextran or electroporationmethods, and retrovirus mediated transfection, as discussed previously,can lead to integration events in the host cell genome that result inoncogene activation or other undesirable consequences. The knowledgethat cationic lipid technology works with messenger RNA is a furtheradvantage to this approach because RNA is turned over rapidly byintracellular nucleases and is not integrated into the host genome. Atransfection system that results in high levels or reversible expressionis preferred to alternative methodology requiring selection andexpansion of stably transformed clones because many of the desiredprimary target cells do not rapidly divide in culture.

The ability to transfect cells at high efficiency with cationicliposomes provides an alternative method for immunization. The gene foran antigen is introduced in to cells which have been removed from ananimal. The transfected cells, now expressing the antigen, arereinjected into the animal where the immune system can respond to the(now) endogenous antigen. The process can possibly be enhanced bycoinjection of either an adjuvant or lymphokines to further stimulatethe lymphoid cells.

Vaccination with nucleic acids containing a gene for an antigen may alsoprovide a way to specifically target the cellular immune response. Cellsexpressing proteins which are secreted will enter the normal antigenprocessing pathways and produce both a humoral and cytotoxic response.The response to proteins which are not secreted is more selective.Non-secreted proteins synthesized in cells expressing only class I MHCmolecules are expected to produce only a cytotoxic vaccination.Expression of the same antigen in cells bearing both class I and classII molecules may produce a more vigorous response by stimulating bothcytotoxic and helper T cells. Enhancement of the immune response mayalso be possible by injecting the gene for the antigen along with apeptide fragment of the antigen. The antigen is presented via class IMHC molecules to the cellular immune system while the peptide ispresented via class II MHC molecules to stimulate helper T cells. In anycase, this method provides a way to stimulate and modulate the immuneresponse in a way which has not previously been possible.

A major disadvantage of subunit vaccines is that glycoprotein antigensare seldom modified correctly in the recombinant expression systems usedto make the antigens. Introducing the gene for a glycoprotein antigenwill insure that the protein product is synthesized, modified andprocessed in the same species and cells that the pathogen protein wouldbe. Thus, the expression of a gene for a human viral glycoprotein willcontain the correct complement of sugar residues. This is importantbecause it has been demonstrated that a substantial component of theneutralizing antibodies in some viral systems are directed atcarbohydrate epitopes.

Any appropriate antigen which is a candidate for an immune response,whether humoral or cellular, can be used in its nucleic acid form. Thesource of the cells could be fibroblasts taken from an individual whichprovide a convenient source of cells expressing only class I MHCmolecules. Alternatively, peripheral blood cells can be rapidly isolatedfrom whole blood to provide a source of cells containing both class Iand class II MHC proteins. They could be further fractionated into Bcells, helper T cells, cytotoxic T cells or macrophage/monocyte cells ifdesired. Bone marrow cells can provide a source of less differentiatedlymphoid cells. In all cases the cell will be transfected either withDNA containing a gene for the antigen or by the appropriate capped andpolyadenylated mRNA transcribed from that gene or a circular RNA,chemically modified RNA, or an RNA which does not require 5′ capping.The choice of the transfecting nucleotide may depend on the duration ofexpression desired. For vaccination purposes, a reversible expression ofthe immunogenic peptide, as occurs on mRNA transfection, is preferred.Transfected cells are injected into the animal and the expressedproteins will be processed and presented to the immune system by thenormal cellular pathways.

Such an approach has been used to produce cytotoxic immunity in modelsystems in mice. Cells lines, malignant continuously growing cells, canbe stably transformed with DNA. When cells are injected into animals,they induce cellular immunity to the expressed antigen. The cationiclipid delivery system will allow this approach to be extended to normal,non-malignant cells taken from a patient.

There are several applications to this approach of targeting cellularimmunity. The first is vaccination against viruses in which antibodiesare known to be required or to enhanced viral infection. There are twostrategies that can be applied here. One can specifically target thecellular pathway during immunization thus eliminating the enhancingantibodies. Alternatively one can vaccinate with the gene for atruncated antigen which eliminate the humoral epitomes which enhanceinfectivity.

The use of DNA or mRNA vaccine therapy could similarly provide a meansto provoke an effective cytotoxic T-cell response to weakly antigenictumors. We propose, for example, that if a tumor-specific antigen wereexpressed by mRNA inside a cell in an already processed form, andincorporated directly into the Class I molecules on the cell surface, acytotoxic T cell response would be elicited.

A second application is that this approach provides a method to treatlatent viral infections. Several viruses (for example, Hepatitis B, HIVand members of the Herpes virus group) can establish latent infectionsin which the virus is maintained intracellularly in an inactive orpartially active form. There are few ways of treating such aninfections. However, by inducing a cytolytic immunity against a latentviral protein, the latently infected cells will be targeted andeliminated.

A related application of this approach is to the treatment of chronicpathogen infections. There are numerous examples of pathogens whichreplicate slowly and spread directly from cell to cell. These infectionsare chronic, in some cases lasting years or decades. Examples of theseare the slow viruses (e.g., Visna), the Scrapie agent and HIV. One caneliminate the infected cells by inducing an cellular response toproteins of the pathogen.

Finally, this approach may also be applicable to the treatment ofmalignant disease. Vaccination to mount a cellular immune response to aprotein specific to the malignant state, be it an activated oncogene, afetal antigen or an activation marker, will result in the elimination ofthese cells.

The use of DNA/mRNA vaccines could in this way greatly enhance theimmunogenicity of certain viral proteins, and cancer-specific antigens,that normally elicit a poor immune response. The mRNA vaccine techniqueshould be applicable to the induction of cytotoxic T cell immunityagainst poorly immunogenic viral proteins from the Herpes viruses,non-A, non-B hepatitis, and HIV, and it would avoid the hazards anddifficulties associated with in vitro propagation of these viruses. Forcell surface antigens, such as viral coat proteins (e.g., HIV gp120),the antigen would be expressed on the surface of the target cell in thecontext of the major histocompatibility complex (MHC), which would beexpected to result in a more appropriate, vigorous and realistic immuneresponse. It is this factor that results in the more efficacious immuneresponses frequently observed with attenuated virus vaccines. Deliveryof a single antigen gene by TGT would be much safer than attenuatedviruses, which can result in a low frequency of disease due toinadequate attenuation.

There is an additional advantage of TGT which can be exploited duringthe vaccine development phase. One of the difficulties with vaccinedevelopment is the requirement to screen different structural variantsof the antigen, for the optimal immune response. If the variant isderived from a recombinant source, the protein usually must be expressedand purified before it can be tested for antigenicity. This is alaborious and time consuming process. With in vitro mutagenesis, it ispossible to obtain and sequence numerous clones of a given antigen. Ifthese antigen can be screened for antigenicity at the DNA or RNA levelby TGT, the vaccine development program could be made to proceed muchfaster.

Finally, in the case of the DNA/mRNA vaccines, the protein antigen isnever exposed directly to serum antibody, but is always produced by thetransfected cells themselves following translation of the mRNA. Hence,anaphylaxis should not be a problem. Thus, the present invention permitsthe patient to be immunized repeatedly without the fear of allergicreactions. The use of the DNA/mRNA vaccines of the present inventionmakes such immunization possible.

One can easily conceive of ways in which this technology can be modifiedto enhance still further the immunogenicity of antigens. T cellimmunization can be augmented by increasing the density of Class I andClass II histocompatibility antigens on the macrophage or other cellsurface and/or by inducing the transfected cell to release cytokinesthat promote lymphocyte proliferation. To this end, one may incorporatein the same liposomes that contain mRNA for the antigen, other mRNAspecies that encode interferons or interleukin-1. These cytokines areknown to enhance macrophage activation. Their systemic use has beenhampered because of side effects. However, when encapsulated in mRNA,along with mRNA for antigen, they should be expressed only by thosecells that co-express antigen. In this situation, the induction of Tcell immunity can be enhanced greatly.

Therapeutic Formulations

Polynucleotide salts: Administration of pharmaceutically acceptablesalts of the polynucleotides described herein is included within thescope of the invention. Such salts may be prepared from pharmaceuticallyacceptable non-toxic bases including organic bases and inorganic bases.Salts derived from inorganic bases include sodium, potassium, lithium,ammonium, calcium, magnesium, and the like. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, basic amino acids, and thelike. For a helpful discussion of pharmaceutical salts, see S. M. Bergeet al., Journal of Pharmaceutical Sciences 66:1-19 (1977) the disclosureof which is hereby incorporated by reference.

Polynucleotides for injection, a preferred route of delivery, may beprepared in unit dosage form in ampules, or in multidose containers. Thepolynucleotidues may be present in such forms as suspensions, solutions,or emulsions in oily or preferably aqueous vehicles. Alternatively, thepolynucleotide salt may be in lyophilized form for reconstitution, atthe time of delivery, with a suitable vehicle, such as sterilepyrogen-free water. Both liquid as well as lyophilized forms that are tobe reconstituted will comprise agents, preferably buffers, in amountsnecessary to suitably adjust the pH of the injected solution. For anyparenteral use, particularly if the formulation is to be administeredintravenously, the total concentration of solutes should be controlledto make the preparation isotonic, hypotonic, or weakly hypertonic.Nonionic materials, such as sugars, are preferred for adjustingtonicity, and sucrose is particularly preferred. Any of these forms mayfurther comprise suitable formulatory agents, such as starch or sugar,glycerol or saline. The compositions per unit dosage, whether liquid orsolid, may contain from 0.1% to 99% of polynucelotide material.

The units dosage ampules or multidose containers, in which thepolynucleoides are packaged prior to use, may comprise an hermeticallysealed container enclosing an amount of polynucleotide or solutioncontaining a polynucleotide suitable for a pharmaceutically effectivedose thereof, or multiples of an effective dose. The polynucleotide ispackaged as a sterile formulation, and the hermetically sealed containeris designed to preserve sterility of the formulation until use.

The container in which the polynucleotide is packaged is labeled, andthe label bears a notice in the form prescribed by a governmentalagency, for example the Food and Drug Administration, which notice isreflective of approval by the agency under Federal law, of themanufacture, use, or sale of the polynucleotide material therein forhuman administration.

Federal law requires that the use of pharmaceutical agents in thetherapy of humans be approved by an agency of the Federal government.Responsibility for enforcement is the responsibility of the Food andDrug Administration, which issues appropriate regulations for securingsuch approval, detailed in 21 U.S.C. 301-392. Regulation for biologicmaterial, comprising products made from the tissues of animals isprovided under 42 U.S.C 262. Similar approval is required by mostforeign countries. Regulations vary from country to country, but theindividual procedures are well known to those in the art.

Dosage and Route of Administration

The dosage to be administered depends to a large extent on the conditionand size of the subject being treated as well as the frequency oftreatment and the route of administration. Regimens for continuingtherapy, including dose and frequency may be guided by the initialresponse and clinical judgment. The parental route of injection into theinterstitial space of tissues is preferred, although other parenteralroutes, such as inhalation of an aerosol formulation, may be required inspecific administration, as for example to the mucous membranes of thenose, throat, bronchial tisues or lungs.

In preferred protocols, a formulation comprising the nakedpolynucleotide in an aqueous carrier is injected into tissue in amountsof from 10 μl per site to about 1 ml per site. The concentration ofpolynucleotide in the formulation is from about 0.1 μg/ml to about 20mg/ml.

Regulation of TGT

Just as DNA based gene transfer protocols require appropriate signalsfor transcribing (promoters, enhancers) and processing (splicingsignals, polyadenylation signals) the mRNA transcript, mRNA based TGTrequires the appropriate structural and sequence elements for efficientand correct translation, together with those elements which will enhancethe stability of the transfected mRNA.

In general, translation efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′UTR) of the RNA. Positive sequence motifs include the translationalinitiation consensus sequence (GCC)^(A)CCATGG (Kozak, Nucleic AcidsRes.15:8125 (1987)) and the 5^(G) 7 methyl GpppG cap structure (Drummondet al., Nucleic Acids Res. 13:7375 (1985)). Negative elements includestable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell48:691(1987)) and AUG sequences or short open reading frames preceded byan appropriate AUG in the 5′ UTR (Kozak, Supra, Rao et al., Mol. andCell. Biol. 8:284(1988)). In addition, certain sequence motifs such asthe beta globin 5′ UTR may act to enhance translation (when placedadjacent to a heterologous 5′ UTR) by an unknown mechanism. There arealso examples of specific 5′ UTR sequences which regulate eukaryotictranslational efficiency in response to environmental signals. Theseinclude the human ferritin 5′ UTR (Hentze et al., Proc. Nat. Acad. Sci.USA 84:6730 (1987)) and the drosophila hsp⁷0 5′ UTR (Klemenz et al.,EMBO Journal 4:2053 (1985)). Finally, there are viral 5′ UTR sequenceswhich are able to bypass normal cap dependant translation andtranslation controls and mediate ann efficient translation of viral orchimeric mRNAs (Dolph et al., J. of Virol. 62:2059 (1988)), Pelletierand Sonnenberg, Nature 334, 320 (1988)). MRNA based TGT protocols musttherefore include appropriate 5′ UTR translational elements flanking thecoding sequence for the protein of interest.

In addition to translational concerns, mRNA stability must be consideredduring the development of mRNA based TGT protocols. As a generalstatement, capping and 3′ polyadenylation are the major positivedeterminants of eukaryotic mRNA stability (Drummond, supra; Ross, Mol.Biol. Med. 5:1(1988)) and function to protect the 5′ and 3′ ends of themRNA from degradation. However, regulatory elements which affect thestability of eukaryotic mRNAs have also been defined, and therefore mustbe considered in the development of mRNA TGT protocols. The most notableand clearly defined of these are the uridine rich 3′ untranslated region(3′ UTR) destabilizer sequences found in many short half-life mRNAs(Shaw and Kamen Cell 46:659 (1986)), although there is evidence thatthese are not the only sequence motifs which result in mRNAdestabilization (Kabnick and Housman, Mol. and Cell. Biol. 8:3244(1988)). In addition, specific regulatory sequences which modulatecellular mRNA half life in response to environmental stimuli have alsobeen demonstrated. These include the estrogen mediated modulation ofVitellogenin mRNA stability (Brock and Shapiro, Cell 34:207 (1983)), theiron dependant regulation of transferrin receptor mRNA stability(Mullner and Kuhn, Cell 53:815 (1988)) which is due to a specific 3′ UTRmotif, the prolactin mediated control of Casein mRNA stability (Guyetteet al., Cell 17:1013 (1989)), the regulation of Fibronectin mRNAstability in response to a number of stimuli (Dean et al., J. Cell.Biol. 106:2159 (1988)), and the control of Histone mRNA stability(Graves et al., Cell 48:615 (1987)). Finally, just as viral RNAsequences have envolved which bypass normal eukaryotic mRNAtranslational controls, likewise some viral RNA sequences seem to beable to confer stability in the absence of 3′ polyadenylation (McGraeand Woodland, Eur. J. of Biochem. 116: 467 (1981)). Some 5′, such asEMC, according to Example 21, are known to function without a cap. Thiscacophony of stability modulating elements must also be carefullyconsidered in developing mRNA based TGT protocols, and can be used tomodulate the effect of an mRNA treatment.

Liposome-forming materials

The science of forming liposomes is now well developed. Liposomes areunilamellar or multilamellar vesicles, having a membrane portion formedof lipophilic material and an interior aqueous portion. The aqueousportion is used in the present invention to contain the polynucleotidematerial to be delivered to the target cell.

It is preferred that the liposome forming materials used herein have acationic group, such as a quaternary ammonium group, and one or morelipophilic groups, such as saturated or unsaturated alkyl groups havingfrom about 6 to about 30 carbon atoms. One group of suitable materialsis described in European Patent Publication No. 0187702. These materialshave the formula:

wherein R¹ and R² are the same or different and are alkyl or alkenyl of6 to 22 carbon atoms, R³, R⁴, and R⁵ are the same or different and arehydrogen, alkyl of 1 to 8 carbons, aryl, aralkyl of 7 to 11 carbons, orwhen two or three of R³, R⁴, and R⁵ are taken together they formquinuclidino, piperidino, pyrrolidino, or morpholino; n is 1 to 8, and Xis a pharmaceutically acceptable anion, such as a halogen. Thesecompounds may be prepared as detailed in the above-identified patentapplication; alternatively, at least one of these compounds,N-(2,3-di-(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimehtylammoniumchloride (DOTMA), is commercially available from Bethesda ResearchLaboratories (BRL), Gaithersburg, Md. 20877, USA.

These quaternary ammonium diether compounds, however, do have somedrawbacks. Because of the ether linkages, they are not readilymetabolized in vivo. When long-term therapy is contemplated, there issome possibility that these materials could accumulate in tissue,ultimately resulting in lipid storage disease and toxic side effects.Accordingly, a preferred class of compositions for use in the presentinvention has the formula:

wherein R¹ and R² are the same or different and are alkyl or alkenyl of5 to 21 carbon atoms, R³, R⁴, and R⁵ are the same or different and arehydrogen, alkyl of 1 to 8 carbons, aryl, aralkyl of 7 to 11 carbons, orwhen two or three of R³, R⁴, and R⁵ are taken together they formquinuclidino, piperidino, pyrrolidino, or morpholino; n is 1 to 8, and Xis a pharmaceutically acceptable anion, such as a halogen. Thesecompounds may be prepared using conventional techniques, such asnucleophilic substitution involving a carboxylic acid and an alkylhalide, by transesterification, or by condensation of an alcohol with anacid or an acid halide.

Moreover, many suitable liposome-forming cationic lipid compounds aredescribed in the literature. See, e.g., L. Stamatatos, et al.,Biochemistry 27:3917-3925 (1988); H. Eibl, et al., Biophysical Chemistry10:261-271 (1979).

Liposome Preparation

Suitable liposomes for use in the present invention are commerciallyavailable. DOTMA liposomes, for example, are available under thetrademark Lipofectin from Bethesda Research Labs, Gaithersburg, Md.

Alternatively, liposomes can be prepared from readily-available orfreshly synthesized starting materials of the type previously described.The preparation of DOTAP liposomes is detailed in Example 6. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P.Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413-7417. Similarmethods can be used to prepare liposomes from other cationic lipidmaterials. Moreover, conventional liposome forming materials can be usedto prepare liposomes having negative charge or neutral charge. Suchmaterials include phosphatidyl choline, cholesterol,phosphatidyl-ethanolamine, and the like. These materials can alsoadvantageously be mixed with the DOTAP or DOTMA starting materials inratios from 0% to about 75%.

Conventional methods can be used to prepare other, noncationicliposomes. These liposomes do not fuse with cell walls as readily ascationic liposomes. However, they are taken up by macrophages in vivo,and are thus particularly effective for delivery of polynucleotide tothese cells. For example, commercially dioleoylphosphatidyl choline(DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15° C. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The present invention is described below in detail using the 23 examplesgiven below; however, the methods described are broadly applicable asdescribed herein and are not intended to be limited by the Examples.

EXAMPLE 1 Preparation of Liposome-forming DOTAP

The cationic liposome-forming material1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) is prepared asreported by L. Stamatatos, et al. (supra) or H. Eibl, et al. (supra).

Briefly, Stamatatos, et al. report that 1 mmol of3-bromo-1,2-propanediol (Aldrich) was acylated for 48 hours at 20° C.with 3 mmol of oleyl chloride (freshly prepared from oleic acid andoxaloyl chloride) in dry, alcohol-free diethyl ether (20 ml) containing5 mmol of dry pyridine. The precipitate of pyridinium hydrochloride wasfiltered off, and the filtrate was concentrated under nitrogen andredissolved in 10 ml of hexane. The hexane solution was washed 3 timeswith an equal volume of 1:1 methanol/0.1 N aqueous NCOONa, pH 3.0, 3times with 1:1 methanol/0.1 N aqueous NaOH, an dl time with 1% aqueousNaCl. The crude 3-bromo-1,2-bis-(oleolyloxy)propane was then stirred for72 hours in a sealed tube with a solution of 15% trimethylamine in drydimethyl sulfoxide (30 ml) at 25° C. The products of this reaction weredissolved in chloroform (200 ml), which was repeatedly washed with 1:1methanol/100 mM aqueous HCOONa, pH 3.0, and then evaporated in vacuo toyield a light yellow oil. This material was purified on a column ofsilicic acid (Bio-Sil A, Bio-Rad Laboratories), eluting with a 0-15%gradient of methanol in chloroform to give the desired product in pureform at 9-10% methanol. The purified product was a colorless, viscousoil that migrates with an R_(f) of 0.4 on thin layer chromatographyplates (silica gel G) that were developed with 50:15:5:5:2CHCl₃/acetone/CH₃OH/CH₃COOH/H₂O.

EXAMPLE 2 Preparation of Plasmids for Making DNA Templates for any Geneof Interest

Suitable template DNA for production of mRNA coding for a desiredpolypeptide may be prepared in accordance with standard recombinant DNAmethodology. As has been previously reported (P. Kreig, et al., NucleicAcids Res. 12:7057-7070 (1984)), a 5′ cap facilitates translation of themRNA. Moreover, the 3′ flanking regions and the poly A tail are believedto increase the half life of the mRNA in vivo.

The readily-available SP6 cloning vector pSP64T provides 5′ and 3′flanking regions from β-globin, an efficiently translated mRNA. Theconstruction of this plasmid is detailed by Kreig, et al. (supra), andis hereby incorporated by this reference. Any cDNA containing aninitiation codon can be introduced into this plasmid, and mRNA can beprepared from the resulting template DNA. This particular plasmid can becut with BglII to insert any desired cDNA coding for a polypeptide ofinterest.

Although good results can be obtained with pSP64T when linearized andthen transcribed in vivo with SP6 RNA polymerase, we prefer to use thexenopus β-globin flanking sequences of pSP64T with phage T7 RNApolymerase. These flanking sequences are purified from pSP64T as thesmall (approx. 150 bp) HindIII to EcoRI fragment. These sequences arethen inserted into a purified linear HindIII/EcoRI fragment (approx. 2.9k bp) from pIBI 31 (commercially available from InternationalBiotechnologies, Inc., Newhaven, Conn. 06535) with T4 DNA ligase.Resulting plasmids, designated pXBG, are screened for orientation andtransformed into E. coli. These plasmids are adapted to receive any geneof interest at a unique BglII restriction site, which is situatedbetween the two xenopus β-globin sequences.

EXAMPLE 3 Preparation of Plasmid Coding for ChloramphenicolAcetyltransferase

A convenient marker gene for demonstrating in vivo expression ofexogenous polynucleotides is chloramphenicol acetyltransferase, CAT. Aplasmid pSP-CAT containing the CAT gene flanked by the xenopus β-globin5′ and 3′ sequences was produced by adding the CAT gene into the BgIIIsite of pSP64T. We used CAT gene in the form of the small BamHI/HindIIIfragment from pSV2-CAT (available from the American Type CultureCollection, Rockville, Md., Accession No. 37155). However, the CAT geneis commonly used in molecular biology and is available from numeroussources. Both the CAT BamHI/HindIII fragment and the BgIII-cleavedpSP64T were incubated with the Klenow fragment to generate blunt ends,and were then ligated with T4 DNA ligase to form pSP-CAT.

The small PstI/HindIII fragment was then generated and purified, whichcomprises the CAT gene between the 5′ and 3′ β-globin flanking sequencesof pSP64T. pIBI31 (International Biotechnologies, Inc.) was cleaved withPstI and HindIII, and the long linear sequence was purified. Thisfragment was then combined with the CAT-gene containing sequence and thefragments were ligated with T4 DNA ligase to form a plasmid designatedpT7CAT An. Clones are selected on the basis of β-galactosidase activitywith Xgal and ampicillin resistance.

EXAMPLE 4 Preparation of Purified DNA Template

The plasmid DNA from Example 3 is grown up and prepared as per Maniatis(supra), except without RNAse, using 2 CsCl spins to remove bacterialRNA. Specifically, E. coli containing pT7CAT An from Example 3 was grownup in ampicillin-containing LB medium. The cells were then pelleted byspinning at 5000 rpm for 10 min. in a Sorvall RC-5 centrifuge (E.I.DuPont, Burbank, Calif. 91510), resuspended in cold TE, pH 8.0,centrifuged again for 10 min. at 5000 rpm., resuspended in a solution of50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA, and 40 mg/ml lysozyme.After incubation for 5 to 10 minutes with occasional inversion, 0.2 NNaOH containing 1% SDS was added, followed after 10 minutes at 0° C.with 3 M potassium acetate and 2 M acetic acid. After 10 more minutes,the material was again centrifuged at 6000 rpm, and the supernatant wasremoved with a pipet. The pellet was then mixed into 0.6 vol.isopropanol (−20° C.), mixed, and stored at −20° C. for 15 minutes. Thematerial was then centrifuged again at 10,000 rpm for 20 min., this timein an HB4 swinging bucket rotor apparatus (DuPont, supra) after whichthe supernatant was removed and the pellet was washed in 70% EtOH anddried at room temperature. Next, the pellet was resuspended in 3.5 mlTE, followed by addition of 3.4 g CsCl and 350 μl of 5 mg/ml EtBr. Theresulting material was placed in a quick seal tube, filled to the topwith mineral oil. The tube was spun for 3.5 hours at 80,000 rpm in aVTi80 centrifuge (Beckman Instruments, Pasadena, Calif., 91051). Theband was removed, and the material was centrifuged again, making up thevolume with 0.95 g CsCl/ml and 0.1 ml or 5 mg/ml EtBr/ml in TE. The EtBrwas then extracted with an equal volume of TE saturated N-Butanol afteradding 3 volumes of TE to the band, discarding the upper phase until theupper phase is clear. Next, 2.5 vol. EtOH was added, and the materialwas precipitated at −20 C for 2 hours. The resultant DNA precipitate isused as a DNA template for preparation of mRNA in vitro.

EXAMPLE 5 Preparation of mRNA for Transfection

The DNA from Example 4 was linearized downstream of the poly A tail witha 5-fold excess of PstI. The linearized DNA was then purified with twophenol/chloroform extractions, followed by two chloroform extractions.DNA was then precipitated with NaOAc (0.3 M) and 2 volumes of EtOH. Thepellet was resuspended at about 1 mg/ml in DEP-treated deionized water.

Next, a transcription buffer was prepared, comprising 400 mM Tris HCl(pH 8.0), 80 mM MgCl₂, 50 mM DTT, and 40 mM spermidine. Then, thefollowing materials were added in order to one volume of DEP-treatedwater at room temperature: 1 volume T7 transcription buffer, preparedabove; rATP, rCTP, and rUTP to 1 mM concentration; rGTP to 0.5 mMconcentration; 7meG(5′)ppp(5′)G cap analog (New England Biolabs,Beverly, Mass. 01951) to 0.5 mM concentration; the linearized DNAtemplate prepared above to 0.5 mg/ml concentration; RNAsin (Promega,Madison, Wis.) to 2000 U/ml concentration; and T7 RNA polymerase (N.E.Biolabs) to 4000 U/ml concentration.

This mixture was incubated for 1 hour at 37 C. The successfultranscription reaction was indicated by increasing cloudiness of thereaction mixture.

Following generation of the mRNA, 2 U RQ1 DNAse (Promega) per microgramof DNA template used was added and was permitted to digest the templatefor 15 minutes. Then, the RNA was extracted twice with chloroform/phenoland twice with chloroform. The supernatant was precipitated with 0.3 MNaOAc in 2 volumes of EtOH, and the pellet was resuspended in 100 μlDEP-treated deionized water per 500 μl transcription product. Thissolution was passed over an RNAse-free Sephadex G50 column (BoehringerMannheim #100 411). The resultant mRNA was sufficiently pure to be usedin transfection of vertebrates in vivo.

EXAMPLE 6 Preparation of Liposomes

A number of liposome preparation methods can be used to advantage in thepractice of the present invention. One particularly preferred liposomeis made from DOTAP as follows:

A solution of 10 mg dioleoyl phosphatidylethanolamine (PE) and 10 mgDOTAP (from Example 1) in 1 ml chloroform is evaporated to dryness undera stream of nitrogen, and residual solvent is removed under vacuumovernight. Liposomes are prepared by resuspending the lipids indeionized water (2 ml) and sonicating to clarity in a closed vial. Thesepreparations are stable for at least 6 months.

Polynucleotide complexes were prepared by mixing 0.5 ml polynucleotidesolution (e.g., from Example 5) at 0.4 mg/ml by slow addition through asyringe with constant gentle vortexing to a 0.5 ml solution of sonicatedDOTMA/PE or DOTAP/PE liposomes at 20 mg/ml, at room temperature. Thisprocedure results in positively charged complexes which willspontaneously deliver the polynucleotide into cells in vivo. Differentratios of positively charged liposome to polynucleotide can be used tosuit the particular need in any particular situation. Alternatively, asreported by Felgner, et al. (supra), it may be advantageous to dilutethe polynucleotide (DNA or RNA) with Hepes buffered saline (150 mM NaCl;20 mM Hepes, pH 7.4) prior to combining the materials to spontaneouslyform liposome/polynucleotide complexes. In many instances, however, theuse of solutions having low ionic strength (such as sucrose) instead ofsaline solution is believed to be preferable; in particular, it isbelieved that such solutions facilitate delivery of polynucleotide tothe cell by minimizing precipitation of polynucleotide/lipid complex.

EXAMPLE 7 In Vivo Expression of Liposomally and Non-liposomallyIntroduced mRNA in the Rat

The ability of mRNA coding for chloramphenicol acetyl transferase (CAT)to transfect cells in vivo and the subsequent expression of the CATprotein was demonstrated by directly injecting 0.200 ml of each of theformulations below, prepared as indicated, into the abdominal muscle ofrats, forming a bleb. Six replicates of each formulation were tested.After 12 to 14 h, the segment of the abdominal muscle into which theinjection was made, weighing approximately 0.1 to 0.2 grams, wasexcised, minced, and placed in a 1.5 ml disposable mortar (Kontes,Morton Grove, Ill.) together with 200 μl of the an aqueous formulationhaving the following components: 20 mM Tris, pH 7.6; 2 mM MgCl₂; and0.1% Triton X-100 surfactant. The contents of the mortar were thenground for 1 minute with a disposable pestle. The mortar was thencovered (with Parafilm) and placed in a 1 liter Parr cell disrupter bomb(Parr Instrument Company, Moline, Ill.) and pressurized to 6 atmosphereswith nitrogen at 4° C. After 30 minutes, the pressure was quicklyreleased to disrupt the tissue and produce a crude lysate. The lysatewas then centrifuged in a microcentrifuge at 13,000 rpm, 4° C., for 10minutes. The supernatant was then decanted and stored at −20° C. untilanalyzed.

The lysates were then assayed for the presence of the CAT protein bythin-layer chromatography. First, 75 μl of each sample (the supernatantprepared above) was incubated for two hours at 37° C. with 5 μl C¹⁴chloramphenicol (Amersham); 20 μl 4 mM Acetyl CoA; and 50 μl 1 M Tris,pH 7.8. Thereafter, 20 μl of 4 mM Acetyl CoA was added, and the mixturewas again incubated for 2 hours at 37° C. The resulting solution wasextracted with 1 ml EtOAc, and the organic phase was removed andlyophilized in a vacuum centrifuge (SpeedVac, Savant Co.). The pelletwas resuspended in 20 μl EtOAc, and was spotted onto a silica gel thinlayer chromatography plate. The plate was developed for 45 minutes in95% chloroform/5% methanol, was dried, and was sprayed with aradioluminescent indicator (Enhance Spray for Surface Radiography, NewEngland Nuclear Corp.). The plate was then sandwiched with Kodak XAR5film with overnight exposure at −70° C., and the film was developed permanufacturer's instructions. The following results were obtained:

mRNA Expression FORMULATION (No. positive/total) 1. 1 ml Optimem; 37.5μg DOTMA 0/6 2. 1 ml Optimem; 15 μg CAT RNA 3/6 3. Formulation 1 plus 15μg CAT RNA 4/6 4. 10% Sucrose; 37.5 μg DOTMA; 15 μg CAT RNA 3/6 5. 10%Sucrose; 187 μg DOTMA; 75 μg CAT RNA 0/6

-   -   Optimem: Serum-free media (Gibco Laboratories, Life        Technologies, Inc, Grand Island, N.Y. 14072)    -   DOTMA: (Lipofectin brand; Bethesda Research Labs, Gaithersburg,        Md.)    -   CAT RNA: From Example 5    -   All formulations made up in DEPC-treated RNAse-free water        (International Biotechnologies, Inc., New Haven, Conn. 06535).

EXAMPLE 8 mRNA Vaccination of Mice to Produce the gp120Protein of HIVVirus

A liposomal formulation containing mRNA coding for the gp120 protein ofthe HIV virus is prepared according to Examples 1 through 5, except thatthe gene for gp120 (pIIIenv3-1 from the Aids Research and ReagentProgram, National Institute of Allergy and Infectious Disease,Rockville, Md. 20852) is inserted into the plasmid pXBG in the procedureof Example 4. A volume of 200 μl of a formulation, prepared according toExample 6, and containing 200 μg/ml of gp120 mRNA and 500 μg/ml 1:1DOTAP/PE in 10% sucrose is injected into the tail vein of mice 3 timesin one day. At about 12 to 14 h after the last injection, a segment ofmuscle is removed from the injection site, and prepared as a cell lysateaccording to Example 7. The HIV specific protein gp120 is identified inthe lysate also according to the procedures of Example 7.

The ability of gp120 antibody present in serum of the mRNA vaccinatedmice to protect against HIV infection is determined by a HT4-6C plaquereduction assay, as follows:

HT4-6C cells (CD4+ HeLa cells) are obtained from Dr. Bruce Chesebro,(Rocky Mountain National Lab, Montana) and grown in culture in RPMImedia (BRL, Gaithersburg, Md.). The group of cells is then divided intobatches. Some of the batches are infected with HIV by addingapproximately 10⁵ to 10⁶ infectious units of HIV to approximately 10⁷HT4-6C cells. Other batches are tested for the protective effect ofgp120 immune serum against HIV infection by adding both the HIV andapproximately 50 μl of serum from a mouse vaccinated with gp120 mRNA.After 3 days of incubation, the cells of all batches are washed, fixedand stained with crystal violet, and the number of plaques counted. Theprotective effect of gp120 immune serum is determined as the reductionin the number of plaques in the batches of cells treated with both gp120mRNA-vaccinated mouse serum and HIV compared to the number in batchestreated with HIV alone.

EXAMPLE 9 mRNA Vaccination of Human Stem Cell-bearing SCID Mice with NEFmRNA Followed by HIV Challenge

Severe combined immunodeficient mice (SCID mice (Molecular BiologyInstitute, (MBI), La Jolla, Calif. 92037)) were reconstituted with adulthuman peripheral blood lymphocytes by injection into the peritonealcavity according to the method of Mosier (Mosier et al., Nature 335:256(1988)). Intraperitoneal injection of 400 to 4000 infectious units ofHIV-1 was then performed. The mice were maintained in a P3 level animalcontainment facility in sealed glove boxes.

MRNA coding for the nef protein if HIV was prepared by obtaining the nefgene in the form of a plasmid (pGM92, from the NIAID, Rockville, Md.20852); removing the nef gene from the plasmid; inserting the nef genein the pXBG plasmid for transcription; and purifying the transcriptionproduct nef mRNA as described in Examples 2 through 5. The nef mRNA wasthen incorporated into a formulation according to Example 6. 200microliter tail vein injections of a 10% sucrose solution containing 200ug/ml NEF RNA and 500 ug/ml 1:1 DOTAP:DOPE (in RNA/liposome complexform) were performed daily on experimental animals, while controlanimals were likewise injected with RNA/liposome complexes containing200 μg/ml yeast tRNA and 500 ug/ml 1:1 DOTAP/DOPE liposomes. At 2, 4 and8 weeks post injection, biopsy specimens were obtained from injectedlymphoid organs and prepared for immunohistochemistry. At the same timepoints, blood samples were obtained and assayed for p24 levels by meansof an ELISA kit (Abbott Labs, Chicago, Ill.) and virus titer by theplaque assay of Example 8. Immunostaining for HIV-1 was performed asdescribed (Namikawa et al., Science 242:1684 (1988)) using polyclonalserum from a HIV infected patient. Positive cells were counted and thenumber of infected cells per high power field (400×) were determined.Using these assays, at least a 2 fold reduction in the number ofpositive staining cells was observed at 8 weeks, and titer and p24expression was reduced by at least 50%. Together, these results indicatea moderate anti-viral effect of the (in vivo) treatment.

A volume of 200 μl of the formulation, containing 200 μg/ml of nef mRNA,and 500 μg/ml 1:1 DOTAP:DOPE in 10% sucrose is injected into the tailvein of the human stem cell-containing SCID mice 3 times in one day.Following immunization, the mice are challenged by infection with aneffective dose of HIV virus. Samples of blood are periodically withdrawnfrom the tail vein and monitored for production of the characteristicHIV protein p24 by an ELISA kit assay (Abbott Labs, Chicago, Ill.).

EXAMPLE 10 A Method of Providing Adenosine Deaminase to Mice by In VivomRNA Transfection

The full-length sequence for the cDNA of the human adenosine deaminase(ADA) gene is obtained from the 1,300 bp EcoRI-AccI fragment of cloneADA 211 (Adrian, G. et al. Mol. Cell Biol. 4:1712 (1984). It isblunt-ended, ligated to BgIII linkers and then digested with BgIII. Themodified fragment is inserted into the BgIII site of pXBG. ADA mRNA istranscribed and purified according to Examples 2 through 5, and purifiedADA mRNA is incorporated into a formulation according to Example 6. Balb3T3 mice are injected directly in the tail vein with 200 μl of thisformulation, containing 200 μg/ml of ADA mRNA, and 500 μg/ml DOTAP in10% sucrose.

The presence of human ADA in the tissues of the liver, skin, and muscleof the mice is confirmed by an isoelectric focusing (IEF) procedure.Tissue extracts were electrofocused between pH 4 and 5 on anon-denaturing gel. The gel was then stained for in situ ADA activity asreported by Valerio, D. et al. Gene 31:137-143 (1984).

A preliminary separation of human and non-human ADA is carried out byfast protein liquid chromatography (FPLC). The proteins are fractionatedon a Pharmacia (Piscataway, N.J.) MonoQ column (HR5γ5) with a lineargradient from 0.05 to 0.5 M KCl, 20 mM Tris (pH 7.5). Activity for ADAwithin the fractions is measured by reacting the fractions with¹⁴C-adenosine (Amersham, Chicago, Ill.) which is converted to inosine.Thin layer chromatography (0.1 M NaPi pH 6.8 saturated ammoniumsulfate:n-propylalcohol/100:60:2) is used to separate the radioactiveinosine from the substrate adenosine.

EXAMPLE 11 In Vivo Expression of Pure RNA and DNA Injected Directly intothe Muscles of Mice

The quadriceps muscles of mice were injected with either 100 μgrams ofpRSVCAT DNA plasmid or 100 μgrams of βgCATβgA_(n) RNA and the muscletissue at the injection site later tested for CAT activity.

Five to six week old female and male Balb/C mice were anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionwas made on the anterior thigh, and the quadriceps muscle was directlyvisualized. The DNA and RNA were injected in 0.1 ml of solution in a 1cc syringe through a 27 gauge needle over one minute, approximately 0.5cm from the distal insertion site of the muscle into the knee and about0.2 cm deep. A suture was placed over the injection site for futurelocalization, and the skin was then closed with stainless steel clips.

3T3 mouse fibroblasts were also transfected in vitro with 20 μg of DNAor RNA complexed with 60 μg of Lipofectin™ (BRL) in 3 ml of Opti-Mem™(Gibco), under optimal conditions described for these cells (Malone, R.et al. Proc. Nat'l. Acad. Sci. USA 86:6077-6081 (1989). The samefibroblasts were also transfected using calcium phosphate according tothe procedure described in Ausubel et al.(Eds) Current Protocols inMolecular Biology, John Wiley and Sons, New York (1989).

The pRSVCAT DNA plasmid and βgCATβgA_(n) RNA were prepared as describedin the preceding examples. The RNA consisted of the chloramphenicolacetyl transferase (CAT) coding sequences flanked by 5′ and 3′ β-globinuntranslated sequences and a 3′ poly-A tract.

Muscle extracts were prepared by excising the entire quadriceps, mincingthe muscle into a 1.5 ml microtube containing 200 μl of a lysis solution(20 mM Tris, pH 7.4, 2 mM MgCl₂ and 0.1% Triton X), and grinding themuscle with a plastic pestle (Kontes) for one minute. In order to ensurecomplete disruption of the muscle cells, the muscle tissue was thenplaced under 600 psi of N₂ in a bomb (Parr) at 4° C. for 15 min beforereleasing the pressure.

Fibroblasts were processed similarly after they were trypsinized off theplates, taken up into media with serum, washed 2× with PBS, and thefinal cell pellet suspended into 200 μl of lysis solution. 75 μl of themuscle and fibroblast extracts were assayed for CAT activity byincubating the reaction mixtures for 2 hours with C¹⁴-chloramphenicol,followed by extraction and thin-layer chromatography, all as describedin Example 7.

Two separate experiments showing CAT activity within extracts of theinjected quadriceps muscles were then performed, and samples from theseexperiments were autoradiogrammed. Among the samples used in suchautoradiograms were control fibroblasts; muscle injected only with 5%sucrose; 0.005 units of non-injected, purified CAT standard; 0.05 unitsof purified CAT (Sigma); muscle injected with 100 micrograms ofBgCATBgA_(n) RNA in 5% sucrose; muscle injected with 100 microgramspRSVCAT DNA in 5% sucrose; 20 micrograms of BgCATBgA_(n) RNA,lipofected, with 60 micrograms of DOTMA, into a 70% confluent 60 mmplate of 3T3 cells (approximately 10⁶); 20 micrograms of pRSVCATlipofected, with 60 micrograms of DOTMA, into a 50% confluent 60 mmplate of 3T3 cells; and 20 micrograms of pRSVCAT calcium phosphatelipofected into a 50% confluent 60 mm plate of 3T3 cells.

CAT activity was readily detected in all four RNA injection sites 18hours after injection and in all six DNA injection sites 48 hours afterinjection. Extracts from two of the four RNA injection sites and fromtwo of the six DNA injection sites contained levels of CAT activitycomparable to the levels of CAT activity obtained from fibroblaststransiently transfected in vitro under optimal conditions. The averagetotal amount of CAT activity expressed in muscle was 960 pg for the RNAinjections and 116 pg for the DNA injections. The variability in CATactivity recovered from different muscle sites probably representsvariability inherent in the injection and extraction technique, sincesignificant variability was observed when pure CAT protein orpRSVCAT-transfected fibroblasts were injected into the muscle sites andimmediately excised for measurement of CAT activity. CAT activity wasalso recovered from abdominal muscle injected with the RNA or DNA CATvectors, indicating that other muscle groups can take up and expresspolynucleotides.

EXAMPLE 12 Site of In Vivo Expression of Pure DNA Injected Directly intothe Muscles of Mice

The site of gene expression in injected muscle was determined byutilizing the pRSVLac-Z DNA vector (P. Norton and J. Coffin Molec. CellBiol. 5:281-290 (1985)) expressing the E. coli β-galactosidase gene forinjection and observing the in situ cytochemical staining of musclecells for E. coli β-galactosidase activity. The quadriceps muscle ofmice was exposed as described in the previous example. Quadricepsmuscles were injected once with 100 μg of pRSVLAC-Z DNA in 20% sucrose.Control muscle was also injected using a solution containing only 20%sucrose. Seven days later the individual quadriceps muscles were removedin their entirety and every fifth 15 μm cross-section washistochemically stained for β-galactosidase activity.

The muscle biopsy was frozen in liquid N₂-cooled isopentane. 15 μmserial sections were sliced using a cryostat and placed immediately ongelatinized slides. The slide were fixed in 1.5% glutaraldehyde in PBSfor 10 minutes and stained 4 hours for β-galactosidase activity (J.Price et al. Proc. Nat'l Acad. Sci. USA 84:156-160 (1987). The musclewas counterstained with eosin.

Approximately 60 muscle cells of the approximately 4000 cells (1.5%)that comprise the entire quadriceps and approximately 10-30% of thecells within the injection area were stained blue. Control muscleinjected with only a 20% sucrose solution did not show any backgroundstaining. Positive β-galactosidase staining within some individualmuscle cells was at least 1.2 mm deep on serial cross-sections, whichmay be the result of either transfection into multiple nuclei or theability of cytoplasmic proteins expressed from one nucleus to bedistributed widely within the muscle cell. Longitudinal sectioning alsorevealed β-galactosidase staining within muscle cells for at least 400μm. In cells adjacent to intensely blue cells, fainter blue stainingoften appeared in their bordering areas. This most likely represents anartifact of the histochemical β-galactosidase stain in which the reactedX-gal product diffuses before precipitating.

Similar results are obtained with linear DNA.

EXAMPLE 13 Dose-response Effects of RNA and DNA Injected into Muscles ofMice

Experiments with the firefly luciferase reporter gene (LUC) explored theeffect of parameters of dose level and time on the total luciferaseextracted from injected muscle.

The RNA and DNA vectors were prepared, and the quadriceps muscles ofmice injected as previously described. Muscle extracts of the entirequadriceps were prepared as described in Example 11, except that thelysis buffer was 100 mM KPi pH 7.8, 1 mM DTT, and 0.1% Triton X. 87.5 μlof the 200 μl extract was analyzed for luciferase activity (J. de Wet etal. Molec. Cell Biol. 7:725-737(1987)) using an LKB 1251 luminometer.Light units were converted to picograms (pg) of luciferase using astandard curve established by measuring the light units produced bypurified firefly luciferase (Analytical Luminescence Laboratory) withincontrol muscle extract. The RNA and DNA preparations prior to injectiondid not contain any contaminating luciferase activity. Control muscleinjected with 20% sucrose had no detectable luciferase activity. All theabove experiments were done two to three times and specifically, the DNAtime points greater than 40 days were done three times.

A. Level of Gene Expression

Varying amounts of BgLUCBgA_(n) RNA in 20% sucrose and pRSVL in 20%sucrose were injected intramuscularly, and 20 micrograms of BgLUCBgA_(n)RNA was lipofected into a million 3T3 fibroblasts. A dose-responseeffect was observed when quadriceps muscles were injected with variousamounts of βgLucβgA_(n) RNA or DNA pRSVL constructs. The injection often times more DNA resulted in luciferase activity increasingapproximately ten-fold from 33 pg luciferase following the injection of10 μg of DNA to 320 pg luciferase following the injection of 100 μg ofDNA. The injection of ten times more RNA also yielded approximately tentimes more luciferase. A million 3T3 mouse fibroblasts in a 60 mm dishwere lipofected with 20 μg of DNA or RNA complexed with 60 μg ofLipofectin™ (Bethesda Research Labs) in 3 ml of Opti-MEM™ (Gibco). Twodays later, the cells were assayed for luciferase activity and theresults from four separate plates were averaged. Twenty μg of pRSVL DNAtransfected into fibroblasts yielded a total of 120 pg of luciferase (6pg luciferase/μg DNA), while 25 μg injected into muscle yielded anaverage of 116 pg of luciferase (4.6 pg luciferase/μg DNA). Theexpression from the RNA vectors was approximately seven-fold moreefficient in transfected fibroblasts than in injected muscles. Twenty μgof βgLucβgA_(n) RNA transfected into fibroblasts yielded a total of 450pg of luciferase, while 25 μg injected into muscle yielded 74 pg ofluciferase. Based on the amount of DNA delivered, the efficiency ofexpression from the DNA vectors was similar in both transfectedfibroblasts and injected muscles.

B. Time Course of Expression

The time course was also investigated. Luciferase activity was assayedat varying times after 25 μg of βgLucβgA_(n) RNA or 100 μg of pRSVL DNAwere injected. Following RNA injection, the average luciferase activityreached a maximum of 74 pg at 18 hours, and then quickly decreased to 2pg at 60 hours. In transfected fibroblasts, the luciferase activity wasmaximal at 8 hours. Following DNA injection into muscle, substantialamounts of luciferase were present for at least 60 days.

The data suggest that luciferase protein and the in vitro RNA transcripthave a half-life of less than 24 hours in muscle. Therefore, thepersistence of luciferase activity for 60 days is not likely to be dueto the stability of luciferase protein or the stability of the in vivoRNA transcript.

EXAMPLE 14 Persistence of DNA in Muscle Following Injection asDetermined by Southern Blot Analysis

Preparations of muscle DNA were obtained from control, uninjectedquadriceps or from quadriceps, 30 days after injection with 100 μg ofpRSVL in 20% sucrose. Two entire quadriceps muscles from the same animalwere pooled, minced into liquid N₂ and ground with a mortar and pestle.Total cellular DNA and HIRT supernatants were prepared (F. M. Ausubel etal.(Eds) Current Protocols in Molecular Biology, John Wiley, New York(1987). Fifteen μg of the total cellular DNA or 10 μl out of the 100 μlof HIRT supernatant were digested, run on a 1.0% agarose gel,transferred to Nytran™ (Schleicher and Schuell, New York), using avacublot apparatus (LKB) and hybridized with multiprimed ³²P-luciferaseprobe (the HindIII-BamHI fragment of pRSVL). Following hybridizationovernight, the final wash of the membrane was with 0.2×SSC containing0.5% SDS at 68° C. Kodak XAR5 film was exposed to the membrane for 45hours at −70° C.

An autoradiogram was then performed on a Southern blot having thefollowing samples: 0.05 ng of undigested pRSVL plasmid; 0.05 ng of BamH1digested pRSVL; BamH1 digest of HIRT supernatant from control muscle;BamH1 digest of cellular DNA from control, uninjected muscle; BamH1digest of HIRT supernatant from two different pools of pRSVL injectedmuscles; BamH1 digest of cellular DNA from two different pools of pRSVLinjected muscle; the same cellular DNA digested with BamH1 and Mbo1; thecellular DNA digested with BamH1 and Dpn1; the cellular DNA digestedwith BglII; and HIRT supernatant digested with BglII. Southern blotanalysis of muscle DNA indicates that the foreign pRSVL DNA is presentwithin the muscle tissue for at least 30 days and is similar to thelevels of DNA present in muscle two and 15 days following injection. Inmuscle DNA digested with BamH1 (which cuts pRSVL once), the presence ofa 5.6 kb band that corresponds to linearized pRSVL suggest that the DNAis present either in a circular, extrachromosomal form or in largetandem repeats of the plasmid integrated into chromosome. In muscle DNAdigested with BgIII (which does not cut pRSVL), the presence of a bandsmaller than 10 kb and at the same size as the open, circular form ofthe plasmid pRSVL implies that the DNA is present extrachromosomally inan open, circular form. The appearance of the pRSVL DNA in HIRTsupernatants and in bacteria rendered ampicillin-resistant followingtransformation with HIRT supernatants also suggest that the DNA ispresent unintegrated. Although the majority of the exogenous DNA appearsto be extrachromosomal, low levels of chromosomal integration cannot bedefinitively excluded. Overexposure of the blobs did not reveal smearsof hybridizing DNA larger than the 10 kb that would represent plasmidDNA integrated at random sites. The sensitivity of the pRSVL DNA ismuscle to DPNI digestion and its resistance to MboI digestion, suggeststhat the DNA has not replicated within the muscle cells.

EXAMPLE 15 In Vivo Expression of Pure DNA Implanted Directly into theMuscle of Mice

pRSVL DNA was precipitated in ethanol and dried. The pellet was pickedup with fine forceps and deposited into various muscle groups asdescribed in the preceding examples. Five days later the muscle wasanalyzed for luciferase activity as described in Example 13. The DNA wasefficiently expressed in different muscle groups as follows:

Implant: Luciferase Activity (Light Units, LU): 25 μg pRSVL DNA ControlBiceps Calf Quadriceps 428 46420 27577 159080 453 53585 34291 35512 1171106865 53397 105176 499 40481

EXAMPLE 16 Direct Gene Delivery into Lung: Intratracheal Injection ofDNA, DNA/CL Complexes or Pure Protein

The DNA luciferase vector (pRSVL), complexed with Lipofectin™, wasinjected intratracheally into rats either in 20% sucrose (2 rats) or in5% sucrose (6 rats). Two days following the injection, the rat lungswere divided into 7 sections: LUL, LLL, RUL, RML, RLL, AL, (defined asfollow) and Trachea. The rat lung differs from that of the human inhaving one large left lung off the left main bronchus. The left lung forthis study was cut in half into a left upper part (LUL) and left lowerpart (LLL). The right lung contains 4 lobes: right cranial lobe (RUL),right middle lobe (RML), right lower lobe ((RLL), and an accessory lobe(AL). Extracts were prepared by mincing these lung parts into separate1.5 ml microtubes containing 200 μl of a lysis solution (20 mM Tris, pH7.4, 2 mM MgCl₂ and 0.1% Triton X), and grinding the lung with a plasticpestle. (Kontes) for one minute. In order to ensure complete disruptionof the lung cells, the lung tissue was then placed under 600 psi of N₂in a Parr bomb at 4° C. for 15 minutes before releasing the pressure.Luciferase assays were done on 87.5 μl of lung extract out of a totalvolume of about 350 μl.

Injection RUL RLL LUL LML LLL AL Trachea Mock 22.6 22.4 21.9 21.3 20.119.8 —  25 μg DNA alone 21.2 21.5 21.8 21.6 21.9 21.2 —  25 μg DNA alone21.7 21.4 21.3 — 22.2 21.5 — 250 μg DNA alone 21.7 23.2 21.9 28.5 22.622.0 21.3 250 μg DNA alone 22.9 22.5 33.3 23.0 25.4 24.3 21.5 250 μg DNAalone 21.8 21.5 21.8 20.4 20.7 20.8 20.7  25 μg DNA/CL 20.8 22.2 19.622.3 22.3 22.0 —  25 μg DNA/CL 22.9 22.0 22.7 21.7 22.8 — 22.18  25 μgDNA/CL 22.2 23.8 22.1 23.9 22.8 — 21.6  25 μg DNA/CL 20.9 20.9 20.9 20.620.3 — 19.3  25 μg DNA/CL 19.8 20.0 20.3 20.2 20.1 20.3 20.1  25 μgDNA/CL 20.5 20.5 19.8 19.5 19.9 19.9 19.8 Luc Protein 105.3 77.1 98.780.0 86.3 89.6 178.9 3 × 10⁴ l.u. Blank 22.5

-   -   Mock: Values are those for an animal that received 25 μg of DNA        in 0.3 ml 20% sucrose into the esophagus. (A sample containing        only water yields 22.5 l.u.)    -   25 μg DNA alone: represent separate animals that received        intratracheal injections of 25 μg of pPGKLuc in 0.3 ml 20%        sucrose.    -   25 μg DNA/CL: represent separate animals that received        intratracheal injections of 25 Mg of pPGKLuc complexed with        Lipofectin™ in 0.3 ml 5% sucrose.    -   The above animals were sacrificed and lung extracts prepared 2        days after injection.    -   Luc Protein 10⁴ l.u.: represents an animal that received the        equivalent of 30,000 light units (l.u.) of purified firefly        luciferase (Sigma), and then was immediately sacrificed.

The luciferase activity in the 25 μg DNA alone and the 25 μg DNA/CLgroups of animals were not greater than that in the mock animal;however, in the 250 μg DNA alone animals, three lung sections showedsmall but reliably elevated l.u. activity above control lung or blanks(Bold, underlined). Duplicate assays on the same extract confirmed theresult. Experience with the LKB 1251 luminometer indicates that thesevalues, although just above background, indicate real luciferaseactivity.

EXAMPLE 17 Luciferase Activity in Mouse Liver Directly Injected with DNAFormulations

The DNA luciferase expression vector pPGKLuc was injectedintrahepatically (IH) into the lower part of the left liver lobe inmice. The pPGKLuc DNA was either injected by itself (450 Mg DNA in 1.0ml 20% sucrose) or complexed with Lipofectin™ (50 μg DNA+150 μgLipofectin™ in 1.0 ml 5% sucrose). Three days following injection, theleft liver lobe was divided into two sections (a lower part where thelobe was injected and an upper part of the lobe distant from theinjection site) and assayed for luciferase activity as described in thepreceding examples.

Luciferase Activity Mice Intrahepatic (Light Units, LU) Liver InjectionLower Upper Blank (20.2 LU) Control: 20% Sucrose Only 20.8 23.8  50 μgpPGKLuc + Lipofectin 35.4 23.1  50 μg pPGKLuc + Lipofectin 38.1 21.4  50μg pPGKLuc + Lipofectin 22.1 22.7 450 μg pPGKLuc 43.7 29.2 450 μgpPGKLuc 78.8 21.7 450 μg pPGKLuc 21.7 20.8

Two of the three animals that received the pure pPGKLuc injections andtwo of the three animals that received pPGKLuc+Lipofectin™ injectionshad luciferase activity significantly above background (bold,underlined). The lower part of the liver lobe, which was directlyinjected, had larger amounts of luciferase activity than the upper part,which was distant from the injection site. Similar results have beenobtained using pRSVCAT DNA expression vector and CAT assays. Luciferaseactivity was not detected three says after similar preparations ofpPGKLuc (+ and − Lipofectin™) were injected into the portal circulationof rats.

EXAMPLE 18 Expression of Growth Hormone Gene Injected into Liver andMuscle

Mice were injected with the pXGH5 (metalothionien promoter-growthhormone fusion gene) (Selden Richard et al., Molec. Cell Biol.6:3173-3179 (1986)) in both liver and muscle. The mice were placed on 76mM zinc sulfate water. Later the animals were bled and the serumanalyzed for growth hormone using the Nichols GH Kit.

-   A. Two mice were injected with 20 μg of pXGH5 gene complexed with 60    μg/ml of Lipofectin in 5% sucrose. One ml of this solution was    injected into the liver and the ventral and dorsal abdominal muscles    were injected with 0.1 ml in 7 sites two times. Two days later, the    animals were bled. The serum of one animal remained at background    level, while that of the other contained 0.75 ng/ml growth hormone.-   B. Three mice were injected with 0.1 ml of 1 mg/ml of pXGH5 in 5%    sucrose, 2× in the quadriceps, 1× in the hamstring muscle, 1× in    pectoralis muscle, and 1× in trapezoid muscles on two separate days.    The results were as follows:

Growth Hormone (ng/ml): Animal No. Day 1 Day 2 1 0.6 0.6 2 0.8 1.0 30.95 0.8 Background: 0.5 ng/ml

EXAMPLE 19 Antibody Production in Mice Directly Injected with a Gene foran Immunizing Peptide

Mice were injected with a quantity of 20 μg of a plasmid constructconsisting of the gp-120 gene, driven by a cytomegalovirus (CMV)promotor. The DNA was injected into the quadriceps muscle of miceaccording to the methods described in Example 11. Mouse 5 was injectedin the quadriceps muscle with 20 μg of plasmid DNA in isotonic sucrose.Mouse 2 was injected with sucrose solution alone. Blood samples wereobtained prior to the injection (Day 0), up to more than 40 days postinjection. The serum from each sample was serially diluted and assayedin a standard ELISA technique assay for the detection of antibody, usingrecombinant gp-120 protein made in yeast as the antigen. Increasedlevels of IgG and IgM antibodies were detected, with the highest levelsbeing detected approximately 5-6 days following injection, tapering offto pre-injection levels approximately 35 days after injection. The studyindicates that the gene retains its signal sequence, and the protein isefficiently excreted from cells.

EXAMPLE 20 Antibody Production in Mice Injected with Cells Transfectedwith a Gene for an Immunizing Peptide

The cell line BALB/C C1.7 (TIB 80) was obtained from the American TypeTissue Culture Collection. These cells were transfected with the gp-120gene construct described in Example 19. To 0.75 ml OptiMEM™ (Gibco.Inc.) were added 6.1 μg of DNA. The quantity of 30 μg of cationicliposomes (containing DOTMA and cholesterol in a 70:30 molar ratio) wereadded to another 0.75 ml OptiMEM™. The mixtures were combined and 1.5 mlof OptiMEM™ containing 20% (v/v) fetal bovine calf serum was added. Thissolution was poured into a 60 mm plastic petri dish containing 80%confluent cells (approximately one million total cells per plate). At3.2 hours after lipofection, the cells were detached from the plate withtrypsin and EDTA treatment, washed with OptiMEM™ and resuspended in 0.1ml OptiMEM™ with 10% fetal calf serum. These cells were injected (IP)into mice. Mouse I2 was injected with the transfected cells. Mouse I1received an identical number of untransfected cells. Blood samples wereobtained prior to the injection (Day 0) and on Days 0, 7 and 14. Theserum samples were processed as in the preceding example. Both IgG andIgM antibodies were detected, with the highest antibody level beingdetected on Day 7.

EXAMPLE 21 Use of Uncapped 5′ Sequences to Direct Translation of DNATransfected into Cells In Vitro

Two different DNA templates were constructed, both of which code for thesynthesis of RNA that express the E. coli. β-galactosidase reportergene. A Lac-Z gene that contains the Kozak consensus sequence wasinserted in place of the luciferase coding sequences of thepβGLucβGA_(n) template to generate the pβGLacZβGA_(n) template. ThepEMCLacZβGA_(n) template was made by replacing the 5′ β-globinuntranslated sequences of pβGLacZβGA_(n) with the 588 bp EcoRl/Ncolfragment from the encephalomyocarditis virus (EMCV). (See constructionof plasmid pE5LVPO from DNA of plasmid pE3T11 in Parks, G. et al., J.Virology 60:376-384, at 378 (1986)). These EMC 5′ untranslated sequenceshad previously been shown to be able to initiate efficient translationin vitro in reticulocyte lysates. We demonstrated that these sequencescan also direct efficient translation when transfected into fibroblastsin culture. The percentage of blue cells was slightly greater in cellstransfected with the uncapped EMCLacZβGA_(n) RNA than in cellstransfected with the capped pEMCLacZβGA_(n) RNA. Transfection witheither uncapped or capped pEMCLacZβGA_(n) RNA yielded a greater numberof positive β-galactosidase cells than transfection with cappedβGLacZβGAn RNA. It has recently been shown that this EMC 5′ untranslatedsequence, as a component of vaccinia-T7 polymerase vectors, can increasetranslation of an uncapped mRNA 4 to 7-fold (Elroy-Stein, O. et al.,Proc. Natl. Acad. Sci. USA 86:6126-6130 (1989)). These EMC sequencesthus have the ability to direct efficient translation from uncappedmessengers.

EXAMPLE 22 T7 Polymerase Transcription in Transfected Cell Cultures

An SV40-T7 polymerase plasmid containing T7 polymerase protein expressedoff the SV40 promotor (Dunn, J. et al., Gene 68: 259 (1988)) wasco-lipofected with the pEMCLacZβGAn template DNA into 3T3 fibroblasts inculture to demonstrate the T7 polymerase transcription can occur viaplasmids. Two different SV40-T7 polymerase expression vectors were used:

-   -   (a) pSV-G1-A: pAR3126-SV40 promotor driving expression of T7        polymerase protein which is directed to the cytoplasm.    -   (b) pSVNU-G1-A: pAR3132-SV40 promotor driving expression of T7        polymerase protein which is directed to the cytoplasm.

Each of these two plasmids were co-lipofected with pEMCLacZβGAn at 1:3and 3:1 ratios into a 60 mm plates of 3T3 cells. The number of blueβ-galactosidase cells were counted and scored as indicated below.

β-gal Ratio: template/ Co-Lipofectant: template polymerase vectorpSV-G1-A pSVKU-G1-A βGlacZβGAn 3:1 0 1 1:3 0 1 EMCLacZβGAn 3:1 74 70 1:345 15

EXAMPLE 23 Expression of Luciferase in Brain Following DirectedInjection of Messenger RNA

Two adult mice and one newborn mouse were injected with the βgLucβgA_(n)mRNA containing the 5′ cap and prepared according to Example 13. In theadult mice, injections were from a stock solution of mRNA at 3.6 μg/μlin 20% sucrose; injection volumes were 5 μl, 2 injections into each ofthe bilateral parietal cortex, 4 injections per mouse. Tissue wasassayed at 18 hours post injection, according to Example 13 using 200 μlof brain homogenate, disrupted in a Parr bomb, and 87.5 μl was taken forassay. The results are as follows:

Hemisphere: Treatment Animal I.D. Left Right Sham Injection AMra 649 629βgLucβgA_(n) AMrb 1,734 1,911The newborn mouse was injected with 1 μl βgLucβgA_(n) (3.6 μg/μl; 20%sucrose) into the bilateral forebrain and tissues were similarlyprocessed and analysed.

Hemisphere: Treatment Animal I.D. Left Right βgLucβgA_(n) NRr 1,569 963

EXAMPLE 24 Functional Expression of Dystrophin in Dystrophic MouseMuscle In Vivo

A plasmid containing the dystrophin gene under control of the RousSarcoma virus promoter was prepared from the Xp21 plasmid containing thecomplete dystrophin coding region and the SV40 poly A segment, which wascloned by Kunkel and colleagues. (Brumeister M., Monaco A P, Gillard EF, van Ommen G J, Affara N A, Ferguson-Smith M A, Kunkel L M, Lehrach,H., “A 10-megabase physical map of human Xp21, including the DuchenneMuscular Dystrophy Gene” Genomics 3:189-202 (1988); Hoffman, E. P. andKunkel, E. M., “Dystrophin abnormalities of Duchenne's/Becher MuscularDystrophy” Neuron 2:1019-1029 (1989); Koenig, M., Monaco, A. P., Kunkel,L. M. “The Complete Sequence of Dystrophin Predicts a Rod-ShapedCytoskeletal Protein” Cell 53(2):219-226 (1988)). 200 μg of the plasmidin 100 μl of phosphate buffered saline was injected into the quadricepsthe mutant mouse strain lacking the dystrophin gene product (MDX mouse;Jackson labs). Expression of functional dystrophin was monitored 7 dayspost injection by immunohistochemistry according to the proceduresdescribed by Watkins et al. and using the same anti-dystrophin antibody(anti-60 kd antibody with a fluorescent secondary antibody) obtainedfrom Kunkel. Functional expression of the dystrophin gene product in thedystrophic mice was detected by comparing the pattern of fluorescenceobserved in cross-sections of quadriceps muscle from injected animals,with the fluorescence pattern observed in normal animals. (Watkins S.C., Hoffman E. P., Slayter H. S., Kinkel L. M., Immunoelectronmicroscopic localization of dystrophin in myofibres. Nature 1988, June30; 333 (6176:863-6). Normal dystrophin expression is localizedunderneath the plasma membrane of the muscle fiber, so that a crosssection of the quadriceps muscle give a fluorescence pattern encirclingthe cell. In addition dystrophin expression was quantitated by Westernblot analysis using the affinity purified anti-60 kd antibody.

EXAMPLE 25 Administration of the Correcting Dystrophin Gene Directlyinto the Muscle of Patients with Duchenne's Muscular Dystrophy

Patients with muscular dystrophy are given multiple 200 ug injections ofplasmid containing the functional dystrophin gene (see previous example)in 100 ul of phosphate buffered saline. While under light anesthesia thepatients are injected at 5 cm intervals into the entire skeletal musclemass directly through the skin without surgery. Patient recoveryevaluated by monitoring twitch tension and maximum voluntarycontraction. In addition, biopsies of 300-500 muscle cells from aninjected area are taken for histological examination, observing musclestructure and biochemical analysis of the presence of dystrophin, whichis absent in patients with Duchenne's muscular dystrophy. Respiratorymuscles, including the intercostal muscles which move the rib cage andthe diaphragm, are particularly important impaired muscle groups inpatients with muscular dystrophy. The intercostals can be reached byinjection through the skin as can the other skeletal muscle groups. Thediaphragm can accessed by a surgical procedure to expose the muscle todirect injection of plasmid DNA.

There will be various modifications, improvements, and applications ofthe disclosed invention that will be apparent to those of skill in theart, and the present application is intended to cover such embodiments.Although the present invention has been described in the context ofcertain preferred embodiments, it is intended that the full scope ofthese be measured by reference to the scope of the following claims.

1. A process for inducing a detectable cellular immune response in amammal, comprising: administering in vivo into tissue of the mammal acomposition comprising a noninfectious, nonintegrating DNA comprising apromoter operably linked to a sequence encoding an immunogen, said DNAbeing complexed to a cationic lipid; wherein said composition isadministered in an amount sufficient that uptake of said DNA into cellsof the mammal occurs, and sufficient expression of said immunogenresults, to induce the detectable cellular immune response.
 2. Theprocess of claim 1, wherein said cationic lipid has a quaternaryammonium group and one or two saturated or unsaturated alkyl groupshaving from about 6 to about 30 carbons.
 3. The process of claim 1,wherein said cationic lipid has a formula selected from the groupconsisting of:

wherein R¹ and R² are the same or different and are C₆ to C₂₂ alkyl orC₆ to C₂₂ alkenyl; R³, R⁴ and R⁵ are the same or different and arehydrogen C₁-C₈ alkyl, aryl, C₇ to C₁₁ aralkyl, or two or three of R³, R⁴and R⁵ can be taken together to form quinuclidino, piperidino,pyrrolidino or morpholino; n is 1 to 8; and X is a pharmaceuticallyacceptable anion; and

 wherein R¹ and R² are the same or different and are C₅ to C₂₁ alkyl orC₅ to C₂₁ alkenyl; R³, R⁴ and R⁵ are the same or different and arehydrogen C₁-C₈ alkyl, aryl, C₇ to C₁₁ aralkyl, or two or three of R³, R⁴and R⁵ can be taken together to form quinuclidino, piperidino,pyrrolidino or morpholino; n is 1 to 8; and X is a pharmaceuticallyacceptable anion.
 4. The process of claim 3, wherein said cationic lipidis selected from the group consisting of:N-(2,3-di-(9-(Z)-octadecenyloxy))prop-1-yl-N,N,N-trimethylammoniumchloride; and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane.
 5. Theprocess of claim 1, wherein said composition further comprises at leastone neutral lipid.
 6. The process of claim 5, wherein said neutral lipidis selected from the group consisting of: phosphatidylcholine,phosphatidylethanolamine, dioleolylphosphatidylcholine,dioleolylphosphatidylglycerol, and dioleolylphosphatidylethanolamine. 7.The process of claim 1, wherein said cationic lipid is selected from thegroup consisting of:N-(2,3-di-(9-(Z)-octadecenyloxy))prop-1-yl-N,N,N-trimethylammoniumchloride; and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane; andwherein said composition further comprises at least one neutral lipidselected from the group consisting of phosphatidylcholine,phosphatidylethanolamine, dioleolylphosphatidylcholine,dioleolylphosphatidylglycerol, and dioleolylphosphatidylethanolamine. 8.The process of claim 7, wherein said composition further comprisescholesterol.
 9. The process of claim 1, wherein said composition furthercomprises cholesterol.
 10. The process of claim 1, wherein said DNA is aplasmid.
 11. The process of claim 1, wherein said promoter is selectedfrom the group consisting of RSV LTR; MPSV LTR; SV40 IEP; CMV IEP; andthe metallothionein promoter.
 12. The process of claim 1, wherein saidimmunogen is a viral protein.
 13. The process of claim 12, wherein saidviral protein is from hepatitis B.
 14. The process of claim 10, whereinsaid composition is administered to said mammal intramuscularly.
 15. Theprocess of claim 10, wherein said composition is administered to saidmammal intravenously.
 16. The process of claim 10, wherein saidcomposition is administered to said mammal subcutaneously.
 17. Theprocess of claim 10, wherein said composition is administered to saidmammal intratumorally.
 18. The process of claim 10, wherein said mammalis a human.
 19. The process of claim 1, wherein said composition isadministered to said mammal intramuscularly.
 20. The process of claim 1,wherein said composition is administered to said mammal intravenously.21. The process of claim 1, wherein said composition is administered tosaid mammal subcutaneously.
 22. The process of claim 1, wherein saidcomposition is administered to said mammal intratumorally.
 23. Theprocess of claim 1, wherein said mammal is a human.
 24. A process fordelivering a biologically active polypeptide to a mammal, comprising:administering in vivo directly into tissue of the mammal a compositioncomprising a noninfectious, nonintegrating DNA comprising a promoteroperably linked to a sequence encoding an immunogen, said DNA beingcomplexed to a cationic lipid; wherein said composition is administeredin an amount sufficient that uptake of said DNA into cells of the mammaloccurs, and sufficient expression results, to produce the polypeptide ata detectable level.
 25. The process of claim 24, wherein said cationiclipid has a quaternary ammonium group and one or two saturated orunsaturated alkyl groups having from about 6 to about 30 carbons. 26.The process of claim 25, wherein said cationic lipid is selected fromthe group consisting of:N-(2,3-di-(9-(Z)-octadecenyloxy))prop-1-yl-N,N,N-trimethylammoniumchloride; and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane.
 27. Theprocess of claim 26, wherein said composition further comprises at leastone neutral lipid selected from the group consisting ofphosphatidylcholine, phosphatidylethanolamine,dioleolylphosphatidylcholine, dioleolylphosphatidylglycerol, anddioleolylphosphatidylethanolamine.
 28. The process of claim 27, whereinsaid composition further comprises cholesterol.
 29. The process of claim24, wherein said DNA is a plasmid.
 30. The process of claim 24, whereinsaid promoter is selected from the group consisting of RSV LTR; MPSVLTR; SV40 IEP; CMV IEP; and the metallothionein promoter.
 31. Theprocess of claim 24, wherein said polypeptide is selected from the groupconsisting of: an interleukin, an interferon, tumor necrosis factor,nerve growth factor, epidermal growth factor, human growth factor,tissue plasmingen activator, factor VIII:C, granulocyte-macrophagecolony-stimulating factor, erythropoietin, insulin, calcitonin, andthymidine kinase.
 32. The process of claim 29, wherein said mammal is ahuman.
 33. The process of claim 24, wherein said composition isadministered to said mammal by a route selected from the groupconsisting of intramuscularly and intrahepatically.
 34. The process ofclaim 29, wherein said composition is administered to said mammal by aroute selected from the group consisting of intramuscularly andintrahepatically.
 35. The process of claim 24, wherein said polypeptideis a cytokine.
 36. The process of claim 35, wherein said cytokine is aninterferon.
 37. The process of claim 24, wherein said mammal is a human.